Compounds and methods for diagnosis and treatment of viral infections

ABSTRACT

Compositions and methods of using these compositions that can include a targeting moiety and a therapeutic agent are described herein. These compositions can be used for diagnosing and/or treating flaviviridae-family viruses including Zika virus, dengue virus, and yellow fever.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to of U.S. Provisional Application No.62/405,780 filed Oct. 7, 2016, the contents of which is incorporated byreference herein in its entirety.

DETAILED DESCRIPTION

Among other things, embodiments of the present invention are directed tocompounds and compositions for targeting dermal macrophages. The presentinvention also provides methods of making such compounds andcompositions. The present invention also provides diagnostic methods andmethods of treatment using compounds comprising a dextran-based moiety,for uses including treating viral infections as disclosed herein.

The invention is not limited in its application to the details ofconstruction and the arrangement of components set forth in thefollowing description or illustrated in the following drawings. Theinvention is capable of other embodiments and of being practiced or ofbeing carried out in various ways.

CD206 is a C-type lectin protein found on macrophages and other cells.In some embodiments, the present invention provides compounds,compositions and methods for the diagnosis and/or treatment of diseasesmediated by CD206-high expressing cells using synthetic macromolecules.These diseases include any condition where macrophages, CD206-highexpressing cells or cells that recognize certain glycolcalyx structuresand their equivalents are involved or recruited, such as those where thenumber of macrophages or other CD206-high and/or lectin-high expressingcells is increased and/or such cells are abnormally localized (e.g., intumors, affected joints, or anatomic regions harboring invadingpathogens, etc.). Such diseases include immune diseases, autoimmunediseases, inflammatory diseases, and infectious diseases. The presentinvention appears, however, to target other receptors found on cellssuch that even non CD206-expressing cells are targeted.

The Virus Genus Flavivirus (Family Flaviviridae—Flavivirus is a genus ofviruses in the family Flaviviridae. This genus includes the West Nilevirus, dengue virus, tick-borne encephalitis virus, yellow fever virus,Zika virus and several other viruses which may cause encephalitis, aswell as insect-specific flaviviruses (ISFs) such as cell fusing agentvirus (CFAV), Palm Creek virus (PCV), and Parramatta River virus (PaRV).

Release Replication Assembly Genus Host Details Tissue Tropism EntryDetails Details Site Site Transmission Flavivirus Humans; Epithelium:skin; Clathrin- Secretion Cytoplasm Cytoplasm Zoonosis; mammals;epithelium: kidney; mediated arthropod bite mosquitoes; epithelium:intestine; endocytosis ticks epithelium: testes macrophages

Flaviviruses have a (+) sense RNA genome and replicate in the cytoplasmof the host cells. The genome mimics the cellular mRNA molecule in allaspects except for the absence of the poly-adenylated (poly-A) tail.This feature allows the virus to exploit cellular apparatuses tosynthesize both structural and non-structural proteins, duringreplication. The cellular ribosome is crucial to the replication of theflavivirus, as it translates the RNA, in a similar fashion to cellularmRNA, resulting in the synthesis of a single polyprotein. In general,the genome encodes 3 structural proteins (Capsid, prM, and Envelope) and8 non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5 andNS5B). The genomic RNA is modified at the 5′ end of positive-strandgenomic RNA with a cap-1 structure (me⁷-GpppA-me²).

Zika Virus—Zika virus (at times, referred to as ZIKV herein; a member ofthe virus family Flaviviridae) can cause Zika virus disease (or, attimes, referred to as Zika fever) and is a member of the virus familyFlaviviridae and the genus Flavivirus. Zika can be spread by Aedesmosquitoes, such as A. aegypti and A. albopictus, which are often activein the day time. Zika virus is related to dengue, yellow fever, Japaneseencephalitis, and West Nile viruses, and other insect vector-spreadviruses. Zika has been known to occur along the equator from Africa toAsia since the 1950s. Over recent years, the virus has spread to westerncontinents including North America and South America.

There is no known specific treatment in the prior art, but paracetamol(acetaminophen) and rest may help with the symptoms but these treatmentshave failed to adequately address the issue. Prior art medications orvaccines have failed to adequately treat Zika. Zika is also especiallyconcerning for pregnant women because the virus can spread from apregnant woman to her fetus. This can result in microcephaly, severebrain malformations, and other birth defects. It has also recently beendiscovered that Zika is neurotropic even in adults. Zika infections inadults may result in temporary paralysis and in Guillain-Barré syndrome,and/or myelitis. Zika can also be sexually transmitted.

In January 2016, the United States Centers for Disease Control andPrevention (CDC) issued travel guidance on affected countries, includingthe use of enhanced precautions, and guidelines for pregnant womenincluding considering postponing travel. As such, needs exist toestablish a way of preventing acquisition of Zika virus.

On Feb. 1, 2016, the World Health Organization declared a Public HealthEmergency of International Concern regarding neurological disordersassociated with the Zika virus (ZIKV) in the Americas. Previously, ZIKVwas a relatively uninvestigated mosquito-borne arbovirus in the genusFlavivirus, family Flaviviridae. ZIKV infections have been known inAfrica and Asia since the 1940s. During the last few years, the viruscaused several outbreaks across Oceania. In May 2015, a ZIKV outbreakwas first reported in Brazil and within months, most countries in LatinAmerica and the Caribbean had reported local transmission of the virus.At present, neither vaccination nor specific antiviral therapies areavailable to prevent/treat ZIKV infections, demonstrating theinternational need for embodiments of the inventions described herein.

Dengue Virus: Dengue fever is a mosquito-borne tropical disease causedby the dengue virus. Symptoms can begin three to fourteen days afterinfection. This may include a high fever, headache, vomiting, muscle andjoint pains, and a characteristic skin rash. Recovery can generallytakes less than two to seven days. In a small proportion of cases, thedisease develops into the life-threatening dengue hemorrhagic fever,resulting in bleeding, low levels of blood platelets and blood plasmaleakage, or into dengue shock syndrome, where dangerously low bloodpressure occurs.

The Dengue virus can be spread by several species of mosquito of theAedes type, principally A. aegypti. The virus has at least fivedifferent types; infection with one type usually gives lifelong immunityto that type, but only short-term immunity to the others. Subsequentinfection with a different type increases the risk of severecomplications. The Dengue diagnosis can be confirmed in a number ofways, including not limited to, detecting antibodies to the virus or itsRNA.

A vaccine for dengue fever has been approved in three countries, but itis not yet commercially available, which is one reason that the presentdisclosure demonstrates that needs exist for the present invention.Prior art treatment of acute dengue is supportive and includes givingfluid either by mouth or intravenously for mild or moderate disease. Formore severe cases blood transfusion may be required. About half amillion people require admission to the hospital per year as a result ofcontracting dengue, and as such, needs exist to prevent and/or treatdengue using compositions and methods described herein. Nonsteroidalanti-inflammatory drug (NSAIDs) such as ibuprofen should not be used toprevent and/or treat dengue.

Dengue has become a global problem since the Second World War and iscommon in more than 110 countries. Each year between 50 and 528 millionpeople are infected and approximately 10,000 to 20,000 die. The earliestdescriptions of an outbreak date from 1779. Aspects of its viral causeand spread were understood by the early 20th century. Apart fromeliminating the mosquitoes, work is ongoing for medication targeteddirectly at the virus, indicating that needs exist for embodiments ofthe invention disclosed herein and that prior art compositions andmethods have failed to adequately achieve the results achieved by thepresent invention.

The characteristic symptoms of dengue are sudden-onset fever, headache(typically located behind the eyes), muscle and joint pains, and a rash.The alternative name for dengue, “breakbone fever”, comes from theassociated muscle and joint pains. The course of infection is dividedinto three phases: febrile, critical, and recovery. The febrile phaseinvolves high fever, potentially over 40° C. (104° F.), and isassociated with generalized pain and a headache; this usually lasts twoto seven days. Nausea and vomiting may also occur. A rash occurs in50-80% of those with symptoms in the first or second day of symptoms asflushed skin, or later in the course of illness (days 4-7), as ameasles-like rash. A rash described as “islands of white in a sea ofred” has also been observed. Some petechiae (small red spots that do notdisappear when the skin is pressed, which are caused by brokencapillaries) can appear at this point, as may some mild bleeding fromthe mucous membranes of the mouth and nose. The fever itself isclassically biphasic or saddleback in nature, breaking and thenreturning for one or two days.

The rash of dengue fever in the acute stage of the infection blancheswhen pressed in some people, the disease proceeds to a critical phase asfever resolves. During this period, there is leakage of plasma from theblood vessels, typically lasting one to two days. This may result influid accumulation in the chest and abdominal cavity as well asdepletion of fluid from the circulation and decreased blood supply tovital organs. There may also be organ dysfunction and severe bleeding,typically from the gastrointestinal tract. Shock (dengue shock syndrome)and hemorrhage (dengue hemorrhagic fever) occur in less than 5% of allcases of dengue, however those who have previously been infected withother serotypes of dengue virus (“secondary infection”) are at anincreased risk. This critical phase, while rare, occurs relatively morecommonly in children and young adults.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C show a series of images of binding of an example of amacrophage-targeting compositions described herein.

FIGS. 2A-2D show a series of images of MR206 expression and binding ofan example of a macrophage-targeting compositions described herein.

FIG. 3 shows that an example of a composition as described herein bindsto, and is internalized by macrophages.

FIG. 4 shows plaque assays showing results of administration of examplesof compositions described herein containing therapeutic agents.

FIG. 5 shows the infectivity reduction of Zika-containing VERO cellsachieved by administration of examples of compounds described hereincontaining therapeutic agents.

FIG. 6 shows the results of administration of examples of compoundsdescribed herein to activated Zika-containing human macrophages.

DEFINITIONS

As used herein, nomenclature for compounds, including organic compounds,can be given using common names, IUPAC, IUBMB, or CAS recommendationsfor nomenclature. When one or more stereochemical features are present,Cahn-Ingold-Prelog rules for stereochemistry can be employed todesignate stereochemical priority, E/Z specification, and the like. Oneof skill in the art can readily ascertain the structure of a compound ifgiven a name, either by systemic reduction of the compound structureusing naming conventions, or by commercially available software, such asCHEMDRAW™ (Perkin Elmer Corporation, U.S.A.).

As used in the specification, the singular forms “a,” “an” and “the”include plural referents unless the context clearly dictates otherwise.Thus, for example, reference to “a functional group,” “an alkyl,” or “aresidue” includes mixtures of two or more such functional groups,alkyls, or residues, and the like.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, a further aspect includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms a further aspect. It willbe further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint. It is also understood that there are a number ofvalues disclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. Forexample, if the value “10” is disclosed, then “about 10” is alsodisclosed. It is also understood that each unit between two particularunits are also disclosed. For example, if 10 and 15 are disclosed, then11, 12, 13, and 14 are also disclosed.

References in the specification to parts by weight of a particularelement or component in a composition denotes the weight relationshipbetween the element or component and any other elements or components inthe composition or article for which a part by weight is expressed.Thus, in a compound containing 2 parts by weight of component X and 5parts by weight component Y, X and Y are present at a weight ratio of2:5, and are present in such ratio regardless of whether additionalcomponents are contained in the compound.

A weight percent (wt. %) of a component, unless specifically stated tothe contrary, is based on the total weight of the formulation orcomposition in which the component is included.

As used herein, the terms “optional” or “optionally” means that thesubsequently described event or circumstance can or cannot occur, andthat the description includes instances where said event or circumstanceoccurs and instances where it does not.

As used herein, the term “subject” can be a vertebrate, such as amammal, a fish, a bird, a reptile, or an amphibian. Thus, the subject ofthe herein disclosed methods can be a human, non-human primate, horse,pig, rabbit, dog, sheep, goat, cow, cat, guinea pig or rodent. The termdoes not denote a particular age or sex. Thus, adult and newbornsubjects, as well as fetuses, whether male or female, are intended to becovered. In one aspect, the subject is a mammal. A patient refers to asubject afflicted with a disease or disorder. The term “patient”includes human and veterinary subjects.

As used herein, the term “treatment” refers to the medical management ofa patient with the intent to cure, ameliorate, stabilize, or prevent adisease, pathological condition, or disorder. This term includes activetreatment, that is, treatment directed specifically toward theimprovement of a disease, pathological condition, or disorder, and alsoincludes causal treatment, that is, treatment directed toward removal ofthe cause of the associated disease, pathological condition, ordisorder. In addition, this term includes palliative treatment, that is,treatment designed for the relief of symptoms rather than the curing ofthe disease, pathological condition, or disorder; preventativetreatment, that is, treatment directed to minimizing or partially orcompletely inhibiting the development of the associated disease,pathological condition, or disorder; and supportive treatment, that is,treatment employed to supplement another specific therapy directedtoward the improvement of the associated disease, pathologicalcondition, or disorder. In various aspects, the term covers anytreatment of a subject, including a mammal (e.g., a human), andincludes: (i) preventing the disease from occurring in a subject thatcan be predisposed to the disease but has not yet been diagnosed ashaving it; (ii) inhibiting the disease, i.e., arresting its development;or (iii) relieving the disease, i.e., causing regression of the disease.In one aspect, the subject is a mammal such as a primate, and, in afurther aspect, the subject is a human. The term “subject” also includesdomesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle,horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mouse,rabbit, rat, guinea pig, fruit fly, etc.).

As used herein, the term “prevent” or “preventing” refers to precluding,averting, obviating, forestalling, stopping, or hindering something fromhappening, especially by advance action. It is understood that wherereduce, inhibit or prevent are used herein, unless specificallyindicated otherwise, the use of the other two words is also expresslydisclosed.

As used herein, the term “diagnosed” means having been subjected to aphysical examination by a person of skill, for example, a physician, andfound to have a condition that can be diagnosed or treated by thecompounds, compositions, or methods disclosed herein.

As used herein, the phrase “identified to be in need of treatment for adisorder,” or the like, refers to selection of a subject based upon needfor treatment of the disorder. For example, a subject can be identifiedas having a need for treatment of a disorder based upon an earlierdiagnosis by a person of skill and thereafter subjected to treatment forthe disorder. It is contemplated that the identification can, in oneaspect, be performed by a person different from the person making thediagnosis. It is also contemplated, in a further aspect, that theidentification can be performed by one who subsequently performed theadministration.

As used herein, the terms “administering” and “administration” refer toany method of providing a pharmaceutical preparation to a subject. Suchmethods are well known to those skilled in the art and include, but arenot limited to, oral administration, transdermal administration,administration by inhalation, nasal administration, topicaladministration, intravaginal administration, ophthalmic administration,intraaural administration, intracerebral administration, rectaladministration, sublingual administration, intradermal administration,buccal administration, and parenteral administration, includinginjectable such as intravenous administration, intraarterialadministration, intramuscular administration, and subcutaneousadministration. Administration can be continuous or intermittent. Invarious aspects, a preparation can be administered therapeutically; thatis, administered to treat an existing disease or condition. In furthervarious aspects, a preparation can be administered prophylactically;that is, administered for prevention of a disease or condition.

The term “contacting” as used herein refers to bringing a disclosedcompound and a cell, a target receptor (e.g. CD206, or other receptor),or other biological entity together in such a manner that the compoundcan affect the activity of the target, either directly; i.e., byinteracting with the target itself, or indirectly; i.e., by interactingwith another molecule, co-factor, factor, or protein on which theactivity of the target is dependent.

As used herein, the terms “effective amount” and “amount effective”refer to an amount that is sufficient to achieve the desired result orto have an effect on an undesired condition. For example, a“therapeutically effective amount” refers to an amount that issufficient to achieve the desired therapeutic result or to have aneffect on undesired symptoms, but is generally insufficient to causeunacceptable adverse side effects. The specific therapeuticallyeffective dose level for any particular patient will depend upon avariety of factors including the disorder being treated and the severityof the disorder; the specific composition employed; the age, bodyweight, general health, sex and diet of the patient; the time ofadministration; the route of administration; the rate of excretion ofthe specific compound employed; the duration of the treatment; drugsused in combination or coincidental with the specific compound employedand like factors well known in the medical arts. For example, it is wellwithin the skill of the art to start doses of a compound at levels lowerthan those required to achieve the desired therapeutic effect and togradually increase the dosage until the desired effect is achieved. Ifdesired, the effective daily dose can be divided into multiple doses forpurposes of administration. Consequently, single dose compositions cancontain such amounts or submultiples thereof to make up the daily dose.The dosage can be adjusted by the individual physician in the event ofany contraindications. Dosage can vary, and can be administered in oneor more dose administrations daily, for one or several days. Guidancecan be found in the literature for appropriate dosages for given classesof pharmaceutical products. In further various aspects, a preparationcan be administered in a “prophylactically effective amount”; that is,an amount effective for prevention of a disease or condition.

The term “pharmaceutically acceptable” describes a material that is notbiologically or otherwise undesirable, i.e., without causing anunacceptable level of undesirable biological effects or interacting in adeleterious manner.

As used herein, the term “pharmaceutically acceptable carrier” refers tosterile aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, as well as sterile powders for reconstitution into sterileinjectable solutions or dispersions just prior to use. Examples ofsuitable aqueous and nonaqueous carriers, diluents, solvents or vehiclesinclude water, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol and the like), carboxymethylcellulose and suitablemixtures thereof, vegetable oils (such as olive oil) and injectableorganic esters such as ethyl oleate. Proper fluidity can be maintained,for example, by the use of coating materials such as lecithin, by themaintenance of the required particle size in the case of dispersions andby the use of surfactants. These compositions can also contain adjuvantssuch as preservatives, wetting agents, emulsifying agents and dispersingagents. Prevention of the action of microorganisms can be ensured by theinclusion of various antibacterial and antifungal agents such asparaben, chlorobutanol, phenol, sorbic acid and the like. It can also bedesirable to include isotonic agents such as sugars, sodium chloride andthe like. Prolonged absorption of the injectable pharmaceutical form canbe brought about by the inclusion of agents, such as aluminummonostearate and gelatin, which delay absorption. Injectable depot formsare made by forming microencapsule matrices of the drug in biodegradablepolymers such as polylactide-polyglycolide, poly(orthoesters) andpoly(anhydrides). Depending upon the ratio of drug to polymer and thenature of the particular polymer employed, the rate of drug release canbe controlled. Depot injectable formulations are also prepared byentrapping the drug in liposomes or microemulsions which are compatiblewith body tissues. The injectable formulations can be sterilized, forexample, by filtration through a bacterial-retaining filter or byincorporating sterilizing agents in the form of sterile solidcompositions which can be dissolved or dispersed in sterile water orother sterile injectable media just prior to use. Suitable inertcarriers can include sugars such as lactose. Desirably, at least 95% byweight of the particles of the active ingredient have an effectiveparticle size in the range of 0.01 to 10 micrometers.

“Alkyl” refers to a saturated aliphatic hydrocarbon including straightchain and branched chain groups. “Alkyl” may be exemplified by groupssuch as methyl, ethyl, n-propyl, isopropyl, n-butyl and the like. Alkylgroups may be substituted or unsubstituted. More than one substituentmay be present. Substituents may also be themselves substituted. Whensubstituted, the substituent group is preferably but not limited toC₁-C₄ alkyl, aryl, heteroaryl, amino, imino, cyano, halogen, alkoxy orhydroxyl. “C₁-C₄ alkyl” refers to alkyl groups containing one to fourcarbon atoms.

“Alkenyl” refers to an unsaturated aliphatic hydrocarbon moietyincluding straight chain and branched chain groups. Alkenyl moietiesmust contain at least one alkene. “Alkenyl” may be exemplified by groupssuch as ethenyl, n-propenyl, isopropenyl, n-butenyl and the like.Alkenyl groups may be substituted or unsubstituted. More than onesubstituent may be present. When substituted, the substituent group ispreferably alkyl, halogen or alkoxy. Substituents may also be themselvessubstituted. Substituents can be placed on the alkene itself and also onthe adjacent member atoms or the alkenyl moiety. “C₂-C₄ alkenyl” refersto alkenyl groups containing two to four carbon atoms.

“Alkynyl” refers to an unsaturated aliphatic hydrocarbon moietyincluding straight chain and branched chain groups. Alkynyl moietiesmust contain at least one alkyne. “Alkynyl” may be exemplified by groupssuch as ethynyl, propynyl, n-butynyl and the like. Alkynyl groups may besubstituted or unsubstituted. More than one substituent may be present.When substituted, the substituent group is preferably alkyl, amino,cyano, halogen, alkoxyl or hydroxyl. Substituents may also be themselvessubstituted. Substituents are not on the alkyne itself but on theadjacent member atoms of the alkynyl moiety. “C₂-C₄ alkynyl” refers toalkynyl groups containing two to four carbon atoms.

“Acyl” or “carbonyl” refers to the group —C(O)R wherein R is alkyl;alkenyl; alkynyl, aryl, heteroaryl, carbocyclic, heterocarbocyclic;C₁-C₄ alkyl aryl or C₁-C₄ alkyl heteroaryl. C₁-C₄ alkylcarbonyl refersto a group wherein the carbonyl moiety is preceded by an alkyl chain of1-4 carbon atoms.

“Alkoxy” refers to the group —O—R wherein R is acyl, alkyl alkenyl,alkyl alkynyl, aryl, carbocyclic; heterocarbocyclic; heteroaryl, C₁-C₄alkyl aryl or C₁-C₄ alkyl heteroaryl.

“Amino” refers to the group —NR′R′ wherein each R′ is, independently,hydrogen, amino, hydroxyl, alkoxyl, alkyl, aryl, cycloalkyl,heterocycloalkyl, heteroaryl, C₁-C₄ alkyl aryl or C₁-C₄ alkylheteroaryl. The two R′ groups may themselves be linked to form a ring.The R′ groups may themselves be further substituted, in which case thegroup also known as guanidinyl is specifically contemplated under theterm ‘amino”.

“Aryl” refers to an aromatic carbocyclic group. “Aryl” may beexemplified by phenyl. The aryl group may be substituted orunsubstituted. More than one substituent may be present. Substituentsmay also be themselves substituted. When substituted, the substituentgroup is preferably but not limited to heteroaryl, acyl, carboxyl,carbonylamino, nitro, amino, cyano, halogen, or hydroxyl.

“Carboxyl” refers to the group —C(═O)O—C₁-C₄ alkyl.

“Carbonyl” refers to the group —C(O)R wherein each R is, independently,hydrogen, alkyl, aryl, cycloalkyl; heterocycloalkyl, heteroaryl, C₁-C₄alkyl aryl or C₁-C₄ alkyl heteroaryl.

“Carbonylamino” refers to the group —C(O)NR′R′ wherein each R′ is,independently, hydrogen, alkyl, aryl, cycloalkyl, heterocycloalkyl,heteroaryl, C₁-C₄ alkyl aryl or C₁-C₄ alkyl heteroaryl. The two R′groups may themselves be linked to form a ring.

“C₁-C₄ alkyl aryl” refers to C₁-C₄ alkyl groups having an arylsubstituent such that the aryl substituent is bonded through an alkylgroup. “C₁-C₄ alkyl aryl” may be exemplified by benzyl.

“C₁-C₄ alkyl heteroaryl” refers to C₁-C₄ alkyl groups having aheteroaryl substituent such that the heteroaryl substituent is bondedthrough an alkyl group.

“Carbocyclic group” or “cycloalkyl” means a monovalent saturated orunsaturated hydrocarbon ring. Carbocyclic groups are monocyclic, or arefused, spiro, or bridged bicyclic ring systems. Monocyclic carbocyclicgroups contain 3 to 10 carbon atoms, preferably 4 to 7 carbon atoms, andmore preferably 5 to 6 carbon atoms in the ring. Bicyclic carbocyclicgroups contain 8 to 12 carbon atoms, preferably 9 to 10 carbon atoms inthe ring. Carbocyclic groups may be substituted or unsubstituted. Morethan one substituent may be present. Substituents may also themselves besubstituted. Preferred carbocyclic groups include cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, and cycloheptyl. Morepreferred carbocyclic groups include cyclopropyl and cyclobutyl. Themost preferred carbocyclic group is cyclopropyl. Carbocyclic groups arenot aromatic.

“Halogen” refers to fluoro, chloro, bromo or iodo moieties. Preferably,the halogen is fluoro, chloro, or bromo.

“Heteroaryl” or “heteroaromatic” refers to a monocyclic or bicyclicaromatic carbocyclic radical having one or more heteroatoms in thecarbocyclic ring. Heteroaryl may be substituted or unsubstituted. Morethan one substituent may be present. When substituted, the substituentsmay themselves be substituted. Preferred but non limiting substituentsare aryl, C₁-C₄ alkylaryl, amino, halogen, hydroxy, cyano, nitro,carboxyl, carbonylamino, or C₁-C₄ alkyl. Preferred heteroaromatic groupsinclude tetrazoyl, triazolyl, thienyl, thiazolyl, purinyl, pyrimidyl,pyridyl, and furanyl. More preferred heteroaromatic groups includebenzothiofuranyl; thienyl, furanyl, tetrazoyl, triazolyl, and pyridyl.

“Heteroatom” means an atom other than carbon in the ring of aheterocyclic group or a heteroaromatic group or the chain of aheterogeneous group. Preferably, heteroatoms are selected from the groupconsisting of nitrogen, sulfur, and oxygen atoms. Groups containing morethan one heteroatom may contain different heteroatoms.

“Heterocarbocyclic group” or “heterocycloalkyl” or “heterocyclic” meansa monovalent saturated or unsaturated hydrocarbon ring containing atleast one heteroatom. Heterocarbocyclic groups are monocyclic, or arefused, spiro, or bridged bicyclic ring systems. Monocyclicheterocarbocyclic groups contain 3 to 10 carbon atoms, preferably 4 to 7carbon atoms, and more preferably 5 to 6 carbon atoms in the ring.Bicyclic heterocarbocyclic groups contain 8 to 12 carbon atoms,preferably 9 to 10 carbon atoms in the ring. Heterocarbocyclic groupsmay be substituted or unsubstituted. More than one substituent may bepresent. Substituents may also be themselves substituted. Preferredheterocarbocyclic groups include epoxy, tetrahydrofuranyl,azacyclopentyl, azacyclohexyl, piperidyl, and homopiperidyl. Morepreferred heterocarbocyclic groups include piperidyl, and homopiperidyl.The most preferred heterocarbocyclic group is piperidyl.Heterocarbocyclic groups are not aromatic.

“Hydroxy” or “hydroxyl” means a chemical entity that consists of —OH.Alcohols contain hydroxy groups. Hydroxy groups may be free orprotected. An alternative name for hydroxy is hydroxyl.

“Leash/leashes” and “linker/linkers” may be used interchangeably herein.The term “leash” or “leashes” can often be used to refer to attachmentmoiety used for a targeting moiety, such as mannose. The term “linker”or “linkers” can be used to refer to the attachment moiety used for atherapeutic agent that may incorporate additional properties related tothe chemistry of the linker and therapeutic agent and the delivery ofthe said agent. Although these terms can be used interchangeably herein,their meaning will be clear to the skilled artisan in view of thecontext with which it is used.

“Member atom” means a carbon, nitrogen, oxygen or sulfur atom. Memberatoms may be substituted up to their normal valence. If substitution isnot specified the substituents required for valency are hydrogen.

“Ring” means a collection of member atoms that are cyclic. Rings may becarbocyclic, aromatic, or heterocyclic or heteroaromatic, and may besubstituted or unsubstituted, and may be saturated or unsaturated. Morethan one substituent may be present. Ring junctions with the main chainmay be fused or spirocyclic. Rings may be monocyclic or bicyclic. Ringscontain at least 3 member atoms and at most 10 member atoms. Monocyclicrings may contain 3 to 7 member atoms and bicyclic rings may containfrom 8 to 12 member atoms. Bicyclic rings themselves may be fused orspirocyclic.

“Thioalkyl” refers to the group —S— alkyl.

“Tilmanocept” refers to a non-radiolabeled precursor of the LYMPHOSEEK®diagnostic agent. Tilmanocept may be a mannosylaminodextran. It can havea dextran backbone to which a plurality of amino-terminated linkers(—O(CH₂)₃S(CH₂)₂NH₂) are attached to the core glucose elements. Inaddition, mannose moieties can be conjugated to amino groups of a numberof the linkers, and the chelator diethylenetriamine pentaacetic acid(DTPA) can be conjugated to the amino group of other linkers notcontaining the mannose. Tilmanocept generally, has a dextran backbone,in which a plurality of the glucose residues comprise anamino-terminated linker:

The mannose moieties are conjugated to the amino groups of the linkervia an amidine linker:

The chelator diethylenetriamine pentaacetic acid (DTPA) is conjugated tothe amino groups the linker via an amide linker:

As described in the prescribing information approved for LYMPHOSEEK® inthe United States, tilmanocept has the chemical name dextran3-[(2-aminoethyl)thio]propyl17-carboxy-10,13,16-tris(carboxymethyl)-8-oxo-4-thia-7,10,13,16-tetraazaheptadec-1-yl3-[[2-[[1-imino-2-(D-mannopyranosylthio)ethyl]amino]ethyl]thio]propylether complexes, has the following molecular formula:[C₆H₁₀O₅]_(n).(C₁₉H₂₈N₄O₉S^(99m)Tc)_(b).(C₁₃H₂₄N₂O₅S₂)_(c).(C₅H₁₁NS)_(a),and contains 3-8 conjugated DTPA molecules; 12-20 conjugated mannosemolecules; and 0-17 amine side chains remaining free. Tilmanocept hasthe following general structure:

Certain of the glucose moieties may have no attached amino-terminatedlinker.

“Sulfonyl” refers to the —S(O)₂R′ group wherein R′ is alkoxy, alkyl,aryl, carbocyclic, heterocarbocyclic; heteroaryl, C₁-C₄ alkyl aryl orC₁-C₄ alkyl heteroaryl.

“Sulfonylamino” refers to the —S(O)₂NR′R′ group wherein each R′ isindependently alkyl, aryl, heteroaryl, C₁-C₄ alkyl aryl or C₁-C₄ alkylheteroaryl.

Compounds described herein can contain one or more double bonds and,thus, potentially give rise to cis/trans (E/Z) isomers, as well as otherconformational isomers. Unless stated to the contrary, the inventionincludes all such possible isomers, as well as mixtures of such isomers.

Unless stated to the contrary, a formula with chemical bonds shown onlyas solid lines and not as wedges or dashed lines contemplates eachpossible isomer, e.g., each enantiomer and diastereomer, and a mixtureof isomers, such as a racemic or scalemic mixture. Compounds describedherein can contain one or more asymmetric centers and, thus, potentiallygive rise to diastereomers and optical isomers. Unless stated to thecontrary, the present invention includes all such possible diastereomersas well as their racemic mixtures, their substantially pure resolvedenantiomers, all possible geometric isomers, and pharmaceuticallyacceptable salts thereof. Mixtures of stereoisomers, as well as isolatedspecific stereoisomers, are also included. During the course of thesynthetic procedures used to prepare such compounds, or in usingracemization or epimerization procedures known to those skilled in theart, the products of such procedures can be a mixture of stereoisomers.

Many organic compounds exist in optically active forms having theability to rotate the plane of plane-polarized light. In describing anoptically active compound, the prefixes D and L or R and S are used todenote the absolute configuration of the molecule about its chiralcenter(s). The prefixes d and 1 or (+) and (−) are employed to designatethe sign of rotation of plane-polarized light by the compound, with (−)or meaning that the compound is levorotatory. A compound prefixed with(+) or d is dextrorotatory. For a given chemical structure, thesecompounds, called stereoisomers, are identical except that they arenon-superimposable mirror images of one another. A specific stereoisomercan also be referred to as an enantiomer, and a mixture of such isomersis often called an enantiomeric mixture. A 50:50 mixture of enantiomersis referred to as a racemic mixture. Many of the compounds describedherein can have one or more chiral centers and therefore can exist indifferent enantiomeric forms. If desired, a chiral carbon can bedesignated with an asterisk (*). When bonds to the chiral carbon aredepicted as straight lines in the disclosed formulas, it is understoodthat both the (R) and (S) configurations of the chiral carbon, and henceboth enantiomers and mixtures thereof, are embraced within the formula.As is used in the art, when it is desired to specify the absoluteconfiguration about a chiral carbon, one of the bonds to the chiralcarbon can be depicted as a wedge (bonds to atoms above the plane) andthe other can be depicted as a series or wedge of short parallel linesis (bonds to atoms below the plane). The Cahn-Inglod-Prelog system canbe used to assign the (R) or (S) configuration to a chiral carbon.

Compounds described herein comprise atoms in both their natural isotopicabundance and in non-natural abundance. The disclosed compounds can beisotopically-labeled or isotopically-substituted compounds identical tothose described, but for the fact that one or more atoms are replaced byan atom having an atomic mass or mass number different from the atomicmass or mass number typically found in nature. Examples of isotopes thatcan be incorporated into compounds of the invention include isotopes ofhydrogen, carbon, nitrogen, oxygen, sulfur, fluorine and chlorine, suchas ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³⁵S, ¹⁸F and ³⁶Cl, respectively.Compounds further comprise prodrugs thereof, and pharmaceuticallyacceptable salts of said compounds or of said prodrugs which contain theaforementioned isotopes and/or other isotopes of other atoms are withinthe scope of this invention. Certain isotopically-labeled compounds ofthe present invention, for example those into which radioactive isotopessuch as ³H and ¹⁴C are incorporated, are useful in drug and/or substratetissue distribution assays. Tritiated, i.e., ³H, and carbon-14, i.e.,¹⁴C, isotopes may be used for their ease of preparation anddetectability. Further, substitution with heavier isotopes such asdeuterium, i.e., ²H, can afford certain therapeutic advantages resultingfrom greater metabolic stability, for example increased in vivohalf-life or reduced dosage requirements and, hence, may be preferred insome circumstances. Isotopically labeled compounds of the presentinvention and prodrugs thereof can generally be prepared by carrying outthe procedures below, by substituting a readily available isotopicallylabeled reagent for a non-isotopically labeled reagent.

It is known that chemical substances form solids which are present indifferent states of order which are termed polymorphic forms ormodifications. The different modifications of a polymorphic substancecan differ greatly in their physical properties. The compounds accordingto the invention can be present in different polymorphic forms, with itbeing possible for particular modifications to be metastable. Unlessstated to the contrary, the invention includes all such possiblepolymorphic forms.

Certain materials, compounds, compositions, and components disclosedherein can be obtained commercially or readily synthesized usingtechniques generally known to those of skill in the art. For example,the starting materials and reagents used in preparing the disclosedcompounds and compositions are either available from commercialsuppliers such as Aldrich Chemical Co., (Milwaukee, Wis.), AcrosOrganics (Morris Plains, N.J.), Fisher Scientific (Pittsburgh, Pa.), orSigma (St. Louis, Mo.) or are prepared by methods known to those skilledin the art following procedures set forth in references such as Fieserand Fieser's Reagents for Organic Synthesis, Volumes 1-17 (John Wileyand Sons, 1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 andSupplemental Volumes (Elsevier Science Publishers, 1989); OrganicReactions, Volumes 1-40 (John Wiley and Sons, 1991); March's AdvancedOrganic Chemistry, (John Wiley and Sons, 4th Edition); and Larock'sComprehensive Organic Transformations (VCH Publishers Inc., 1989).

Unless otherwise expressly stated, it is in no way intended that anymethod set forth herein be construed as requiring that its steps beperformed in a specific order. Accordingly, where a method claim doesnot actually recite an order to be followed by its steps or it is nototherwise specifically stated in the claims or descriptions that thesteps are to be limited to a specific order, it is no way intended thatan order be inferred, in any respect. This holds for any possiblenon-express basis for interpretation, including: matters of logic withrespect to arrangement of steps or operational flow; plain meaningderived from grammatical organization or punctuation; and the number ortype of embodiments described in the specification.

Disclosed are the components to be used to prepare the compositions ofthe invention as well as the compositions themselves to be used withinthe methods disclosed herein. These and other materials are disclosedherein, and it is understood that when combinations, subsets,interactions, groups, etc. of these materials are disclosed that whilespecific reference of each various individual and collectivecombinations and permutation of these compounds cannot be explicitlydisclosed, each is specifically contemplated and described herein. Forexample, if a particular compound is disclosed and discussed and anumber of modifications that can be made to a number of moleculesincluding the compounds are discussed, specifically contemplated is eachand every combination and permutation of the compound and themodifications that are possible unless specifically indicated to thecontrary. Thus, if a class of molecules A, B, and C are disclosed aswell as a class of molecules D, E, and F and an example of a combinationmolecule, A-D is disclosed, then even if each is not individuallyrecited each is individually and collectively contemplated meaningcombinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considereddisclosed. Likewise, any subset or combination of these is alsodisclosed. Thus, for example, the sub-group of A-E, B-F, and C-E wouldbe considered disclosed. This concept applies to all aspects of thisapplication including, but not limited to, steps in methods of makingand using the compositions of the invention. Thus, if there are avariety of additional steps that can be performed it is understood thateach of these additional steps can be performed with any specificembodiment or combination of embodiments of the methods of theinvention.

It is understood that the compositions disclosed herein have certainfunctions. Disclosed herein are certain structural requirements forperforming the disclosed functions, and it is understood that there area variety of structures that can perform the same function that arerelated to the disclosed structures, and that these structures willtypically achieve the same result.

Compounds

Embodiments of the present invention can employ a carrier constructcomprising a polymeric (e.g., carbohydrate) backbone that can comprise aCD206 targeting moiety attached thereto (e.g., mannose) to deliver oneor more active pharmaceutical ingredients. Examples of such constructsinclude mannosylamino dextrans (MAD), which can comprise a dextranbackbone having conjugated to glucose residues of the backbone mannosemolecules and having conjugated to other glucose residues of thebackbone an active pharmaceutical ingredient. Tilmanocept is a specificexample of a MAD. A tilmanocept derivative that is tilmanocept withoutDTPA conjugated thereto is a further example of a MAD (sometimesreferred to as mtilmanocept).

In some embodiments, the present invention provides a compoundcomprising a dextran-based moiety or backbone having one or more CD206targeting moieties and one or more therapeutic agents attached thereto.The dextran-based moiety generally comprises a dextran backbone similarto that described in U.S. Pat. No. 6,409,990 (the '990 patent), which isincorporated herein by reference in its entirety. Thus, the backbonecomprises a plurality of glucose moieties (i.e., residues) primarilylinked by α-1,6 glycosidic bonds. Other linkages such as α-1,4 and/orα-1,3 bonds may also be present. In some embodiments, not every backbonemoiety is substituted. In some embodiments, CD206 targeting moieties areattached to between about 10% and about 50% of the glucose residues ofthe dextran backbone, or between about 20% and about 45% of the glucoseresidues, or between about 25% and about 40% of the glucose residues. Insome embodiments, every three glucose residues may be substituted. Insome embodiments, every four glucose residues may be substituted. Insome embodiments, every five glucose residues may be substituted. Someembodiments may comprise one mannose positioned on every third glucoseresidue. Some embodiments may comprise one mannose positioned on everyfourth glucose residue. Some embodiments may comprise one mannosepositioned on every fifth glucose residue. In some embodiments, thedextran-based moiety is about 50-100 kilodaltons (kDa). Thedextran-based moiety may be at least about 50 kDa, at least about 60kDa, at least about 70 kDa, at least about 80 kDa, or at least about 90kDa. The dextran-based moiety may be less than about 100 kDa, less thanabout 90 kDa, less than about 80 kDa, less than about 70 kDa, or lessthan about 60 kDa. In some embodiments, the dextran backbone has amolecular weight (MW) of between about 1 and about 50 kDa, while inother embodiments the dextran backbone can have a MW of between about 5and about 25 kDa. In embodiments, the dextran backbone can have a MW ofbetween about 8 and about 15 kDa, such as about 10 kDa. While in otherembodiments the dextran backbone can have a MW of between about 1 andabout 5 kDa, such as about 2 kDa. Certain embodiments of compositionscan comprise a backbone that is between about 1 to about 5 kDa, about 1to about 10 kDa, about 1 to about 15 kDa, about 5 to about 12 kDa, about5 to about 10 kDa, and ranges therebetween. In some embodiments, acomposition may comprise between about 3 to about 7 mannose molecules,about 5 to about 10 mannose molecules, about 10 to about 15 mannosemolecules, about 15 to about 20 mannose molecules, about 16 to about 17mannose molecules, and ranges therebetween. In some embodiments, abackbone may be about 1 to about 3 kDa and may further comprise about 3to about 7 mannose molecules. In some embodiments, a backbone may beabout 10 kDa and may further comprise about 15 to about to about 20, orabout 16 to about 17 mannose molecules. An embodiment may comprise abackbone that is about 10 kDa and further comprise about 2 therapeuticagent mole-cules, wherein the therapeutic agent molecules can comprisedoxorubicin, and further comprise about 16 to about 17 mannosemolecules. Such a configuration has unexpectedly superior and improvedsolubility, improved clarity, improved injectability and distribution,and can reduce the amount of active drug required to achieve atherapeutic result. Some embodiments may comprise therapeutic agentswherein the compositions comprises between about 1 and about 5therapeutic agent molecules.

Some embodiments may comprise a backbone that is not a dextran backbone.Some embodiments may have a monosaccharide-based backbone that does notcomprise dextran. The backbone of a carbohydrate-based carrier moleculesdescribed herein can comprise a glycan other than dextran, wherein theglycan comprises a plurality of monosaccharide residues (i.e., sugarresidues or modified sugar residues). In certain embodiments, the glycanbackbone has sufficient monosaccharide residues, as well as optionalgroups such as one or more amino acids, polypeptides and/or lipids, toprovide a MW of about 1 to about 50 kDa. As would be appreciated by theskilled artisan when considering the disclosure contained herein, whenreferring to a “dextran” backbone, other monosaccharide residues may beconsidered to be substituted in compounds described herein. Additionaldescriptions of carbohydrate-backbone-based carrier molecules used fortargeting CD206 are described in PCT application No. US/2017/055211,which is herein incorporated by reference in its entirety.

Certain embodiments of compositions can comprise a backbone that isbetween about 1 to about 5 kDa, about 1 to about 10 kDa, about 1 toabout 15 kDa, about 5 to about 12 kDa, about 5 to about 10 kDa, andranges there between. In some embodiments, a composition may comprisebetween about 2 to about 7 mannose molecules, about 5 to about 10mannose molecules, about 10 to about 15 mannose molecules, about 15 toabout 28 mannose molecules, about 16 to about 17 mannose molecules, andranges there between. In some embodiments, a backbone may be about 1 toabout 3 kDa and may further comprise about 3 to about 7 mannosemolecules. In some embodiments, a backbone may be about 10 kDa and mayfurther comprise about 15 to about to about 20, or about 16 to about 17mannose molecules. In some embodiments, a therapeutic molecule cancomprise about 1 to about 5 molecules (e.g., therapeutic agents), about3 to 10 molecules, about 7 to about 20 molecules, and rangestherebetween. An embodiment may comprise a backbone that is about 10 kDaand further comprise about 2 therapeutic agent molecules, wherein thetherapeutic agent molecules can comprise doxorubicin, and furthercomprise about 16 to about 17 mannose molecules. Such a configurationhas unexpectedly superior and improved solubility, improved clarity,improved injectability and biodistribution, and can reduce the amount ofactive drug required to achieve a therapeutic result at a fraction ofthe free therapeutic agent.

Carrier molecules having smaller MW dextran backbones may be appropriatefor instances where the molecule is desired to cross the blood-brainbarrier, or when reduced residence time is desired (i.e., the durationof binding to CD206 is reduced). Carrier molecules having larger MWdextran backbones may be appropriate for instances where increasedresidence time is desired (i.e., the duration of circulation andexposure to CD206 is increased). In certain embodiments, carriermolecules having smaller MW dextran backbones (e.g., about 1 to about 5kDa) may be employed when more efficient receptor substrates areattached to the dextran backbone (e.g., branched mannose moieties, asdescribed below). More efficient receptor substrates can bind to CD206for longer durations and/or more effectively, thus allowing for the useof smaller dextran backbones.

In some embodiments, the CD206 targeting moiety is selected from, butnot limited to, mannose, fucose, galactose, n-acetylgalactosamine, andn-acetylglucosamine and combinations of these. In some embodiments, thetargeting moieties are attached to between about 10% and about 50% ofthe glucose residues of the dextran backbone, or between about 20% andabout 45% of the glucose residues, or between about 25% and about 40% ofthe glucose residues. (It should be noted that the MWs referencedherein, as well as the number and degree of conjugation of receptorsubstrates, linkers, and diagnostic/therapeutic moieties attached to thedextran backbone refer to average amounts for a given quantity ofcarrier molecules, since the synthesis techniques will result in somevariability.)

In some embodiments, the one or more CD206 targeting moieties and one ormore therapeutic agents (or drugs) and/or detection labels are attachedto the dextran-based moiety through a linker. The linker may be attachedat from about 50% to about 100% of the backbone moieties or about 70% toabout 90%. In embodiments with multiple linkers, the linkers may be thesame or different. In some embodiments, the linker is anamino-terminated linker. In some embodiments, the linkers may comprise—O(CH₂)₃S(CH₂)₂NH—. In some embodiments, the linker may be a chain offrom 1 to 20 member atoms selected from carbon, oxygen, sulfur, nitrogenand phosphorus. The linker may be a straight chain or branched. Thelinker may also be substituted with one or more substituents including,but not limited to, halo groups, perfluoroalkyl groups, perfluoroalkoxygroups, alkyl groups, such C₁₋₄ alkyl, alkenyl groups, such as C₁₋₄alkenyl, alkynyl groups, such as C₁₋₄ alkynyl, hydroxy groups, oxogroups, mercapto groups, alkylthio groups, alkoxy groups, nitro groups,azidealkyl groups, aryl or heteroaryl groups, aryloxy or heteroaryloxygroups, aralkyl or heteroaralkyl groups, aralkoxy or heteroaralkoxygroups, HO—(C═O)—groups, heterocylic groups, cycloalkyl groups, aminogroups, alkyl- and dialkylamino groups, carbamoyl groups, alkylcarbonylgroups, alkylcarbonyloxy groups, alkoxycarbonyl groups,alkylaminocarbonyl groups, dialkylamino carbonyl groups, arylcarbonylgroups, aryloxycarbonyl groups, alkyl sulfonyl groups, arylsulfonylgroups, —NH—NH₂; ═N—H; ═N-alkyl; —SH; —S-alkyl; —NH—C(O)—; —NH—C(═N)—and the like. Other suitable linkers would be known to one of ordinaryskill in the art.

In some embodiments, the one or more therapeutic agent is attached via abiodegradable linker. In some embodiments, the biodegradable linkercomprises an acid sensitive, such as a hydrazone moiety. The use of anacid sensitive linker enables the drug to be transported into the celland allows for the release of the drug substantially inside of the cell.In certain embodiments, the linker comprises a biodegradable moietyattached to a linker.

Various other linkers known to those skilled in the art or subsequentlydiscovered may be used in place of (or in addition to)—O(CH₂)₃S(CH₂)₂NH₂. These include, for example, bifunctional linkergroups such as alkylene diamines (H₂N—(CH₂)_(r)—NH₂), where r is from 2to 12; aminoalcohols (HO—(CH₂)_(r)—NH₂), where r is from 2 to 12;aminothiols (HS—(CH₂)_(r)—NH₂), where r is from 2 to 12; amino acidsthat are optionally carboxy-protected; ethylene and polyethylene glycols(H—(O—CH₂—CH₂)_(n)—OH, where n is 1-4). Suitable bifunctional diaminesinclude ethylenediamine, 1,3-propanediamine, 1,4-butanediamine,spermidine, 2,4-diaminobutyric acid, lysine, 3,3′-diaminodipropylamine,diaminopropionic acid, N-(2-aminoethyl)-1,3-propanediamine,2-(4-aminophenyl)ethylamine, and similar compounds. One or more aminoacids also can be employed as the bifunctional linker molecule, such asβ-alanine, γ-aminobutyric acid or cysteine, or an oligopeptide, such asdi- or tri-alanine.

Other bifunctional linkers include:

where r is from 2-5,

where r is from 2-5,

where is from 1-5,

where is from 2-5,

where is from 1-5,

The therapeutic agent can include any compound known to be useful forthe treatment of a disease vectored by biting insects, such as Zikavirus, dengue virus, and yellow fever. Therapeutic agents include, butare not limited to, chemotherapeutic agents, such as doxorubicin;dexamethasone; anti-infective agents, such as antibiotics (e.g.tetracycline, streptomycin, amphotericin and isoniazid), heavy metalssuch as antimony (e.g. pentavalent antimonials), anti-virals,anti-fungals, and anti-parasitics; immunological adjuvants; steroids;nucleotides, such as DNA, RNA, RNAi, siRNA, CpG or Poly (I:C); peptides;proteins; or metals such as silver, gallium or gadolinium, paromomycin,miltefosine, fluconazole, pentamide, Meglumine antimoniate, andcombinations thereof.

In certain embodiments, the therapeutic agent is an antimicrobial drugselected from the group comprising: an antibiotic; an anti-tuberculosisantibiotic (such as isoniazid, streptamycin, or ethambutol); drugs witheffect on Zika virus; drugs with effect on Dengue virus, drugs witheffect on family Flaviviridae viruses; an anti-viral or anti-retroviraldrug, for example an inhibitor of reverse transcription (such aszidovudin) or a protease inhibitor (such as indinavir). In certainembodiments, the therapeutic agent is an anti-microbial active, such asamoxicillin, ampicillin, tetracyclines, aminoglycosides (e.g.,streptomycin), macrolides (e.g., erythromycin and its relatives),chloramphenicol, ivermectin, rifamycins and polypeptide antibiotics(e.g., polymyxin, bacitracin) and zwittermicin. In certain embodiments,the therapeutic agent is selected from isoniazid, doxorubicin,streptomycin, and tetracycline.

In some embodiments, the therapeutic agent comprises a high energykilling isotope which has the ability to kill macrophages and tissue inthe surrounding macrophage environment. Suitable radioisotopes include:^(210/212/213/214)Bi, ^(131/140)Ba, ^(11/14)C, ⁵¹Cr, ^(67/68)Ga, ¹⁵³Gd,^(99m)Tc, ^(88/90/91)Y, ^(123/124/125/131)I, ^(111/115m)In, ¹⁸F, ¹⁰⁵Rh,¹⁵³Sm, ⁶⁷Cu, ¹⁶⁶Ho, ¹⁷⁷Lu, ^(186/188)Re, ^(32/33)P, ^(46/47)Sc,^(72/75)Se, ³⁵S, ¹⁸²Ta, ^(123m/127/129/132)Te, ⁶⁵Zn and ^(89/95)Zr.

In embodiments, a therapeutic agent comprises a non-radioactive speciesselected from, but not limited to, the group consisting of: Bi, Ba, Mg,Ni, Au, Ag, V, Co, Pt, W, Ti, Al, Si, Os, Sn, Br, Mn, Mo, Li, Sb, F, Cr,Ga, Gd, I, Rh, Cu, Fe, P, Se, S, Zn and Zr.

In some embodiments, a therapeutic agent is selected from the groupconsisting of cytostatic agents, alkylating agents, antimetabolites,anti-proliferative agents, tubulin binding agents, hormones and hormoneantagonists, anthracycline drugs, vinca drugs, mitomycins, bleomycins,cytotoxic nucleosides, pteridine drugs, diynenes, podophyllotoxins,toxic enzymes, and radiosensitizing drugs. By way of more specificexample, the therapeutic agent is selected from the group consisting ofmechlorethamine, triethylenephosphoramide, cyclophosphamide, ifosfamide,chlorambucil, busulfan, melphalan, triaziquone, nitrosourea compounds,adriamycin, carminomycin, daunorubicin (daunomycin), doxorubicin,isoniazid, indomethacin, gallium(III), 68gallium(III), aminopterin,methotrexate, methopterin, mithramycin, streptonigrin,dichloromethotrexate, mitomycin C, actinomycin-D, porfiromycin,5-fluorouracil, floxuridine, ftorafur, 6-mercaptopurine, cytarabine,cytosine arabinoside, podophyllotoxin, etoposide, etoposide phosphate,melphalan, vinblastine, vincristine, leurosidine, vindesine, leurosine,taxol, taxane, cytochalasin B, gramicidin D, ethidium bromide, emetine,tenoposide, colchicin, dihydroxy anthracin dione, mitoxantrone,procaine, tetracaine, lidocaine, propranolol, puromycin, ricin subunitA, abrin, diptheria toxin, botulinum, cyanginosins, saxitoxin,shigatoxin, tetanus, tetrodotoxin, trichothecene, verrucologen,corticosteroids, progestins, estrogens, antiestrogens, androgens,aromatase inhibitors, calicheamicin, esperamicins, and dynemicins andcombinations thereof.

In embodiments wherein the therapeutic agent is a hormone or hormoneantagonist, the therapeutic agent may be selected from the groupconsisting of prednisone, hydroxyprogesterone, medroprogesterone,diethylstilbestrol, tamoxifen, testosterone, and aminogluthetimide andcombinations thereof.

In embodiments wherein the therapeutic agent is a prodrug, thetherapeutic agent may be selected from the group consisting ofphosphate-containing prodrugs, thiophosphate-containing prodrugs,sulfate containing prodrugs, peptide containing prodrugs,(-lactam-containing prodrugs, optionally substitutedphenoxyacetamide-containing prodrugs, optionally substitutedphenylacetamide-containing prodrugs, 5-fluorocytosinem, and5-fluorouridine prodrugs that can be converted to the more activecytotoxic free drug and combinations thereof.

Examples of constructs useful in the present invention includemannosylamino dextrans (MAD) such as tilmanocept and m-tilmanocept. Insome embodiments, the dextran-based moiety having at least one CD206targeting moiety attached thereto can be a compound of Formula

wherein the * indicates the point at which the therapeutic agent can beattached. In certain embodiments, the therapeutic agent can be attachedvia a linker. In certain embodiments, x can be between about 10 to about25, about 5 to about 25, about 10 to about 20, about 15 to about 25,about 15 to about 20 and ranges therebetween. In some embodiments, y canbe between about 35 and about 70, about 40 and about 70, about 50 andabout 65, and ranges therebetween. In some embodiments, z can be betweenabout 40 to about 70, about 50 to about 65, about 50 to about 60 andranges therebetween.

In other embodiments, the compound of the present invention can be acompound of Formula (II):

Whereineach X is independently H, L₁-A, or L₂-R; each L₁ and L₂ areindependently linkers;each A independently comprises a therapeutic agent or a detection labelor H;each R independently comprises a CD206 targeting moiety or H; andn is an integer greater than zero.

In certain embodiments, L₁ is a linker as described above. In certainembodiments, L₂ is a linker as described above.

Synthesis

The compounds of this invention can be prepared by employing reactionsas shown in the disclosed schemes, in addition to other standardmanipulations that are known in the literature, exemplified in theexperimental sections or clear to one skilled in the art. The followingexamples are provided so that the invention might be more fullyunderstood, are illustrative only, and should not be construed aslimiting. For clarity, examples having fewer substituents can be shownwhere multiple substituents are allowed under the definitions disclosedherein.

It is contemplated that each disclosed method can further compriseadditional steps, manipulations, and/or components. It is alsocontemplated that any one or more step, manipulation, and/or componentcan be optionally omitted from the invention. It is understood that adisclosed method can be used to provide the disclosed compounds. It isalso understood that the products of the disclosed methods can beemployed in the disclosed compositions, kits, and uses.

The compounds of the present invention may be synthesized by any numberof ways known to one of ordinary skill in the art. For example, linker 2can be synthesized by opening succinic anhydride ring by tert-butylcarbazate. The resulting carboxylic acid is converted to thecorresponding N-hydroxy succinimide (NHS) ester using EDC couplingreagent. MAD is then functionalized with linker 2 by forming an amidelinkage. Then, the Boc protecting group can be removed under diluteacidic condition (typically 30-40% trifluoroacetic acid in DMSO) toobtain 4. Dilute acidic condition is required to avoid any unwantedcleavage of the glycosidic linkage present in dextran backbone. Theresulting functionalized MAD can be purified by size exclusionfiltration.

Alternatively, compounds according to the present invention may besynthesized according to Scheme 2. Free primary amine groups of MAD canbe reacted with an excess of lactone under anhydrous condition.Unreacted lactone can be removed under reduced pressure to obtainmodified MAD 6. The corresponding hydrazine derivative 7 can be preparedby reductive amination reaction using sodium cyanoborohydride or sodiumtriacetoxy borohydride as the reducing agent.

The conjugation of oxo-containing therapeutic agents to MAD derivatives4 or 7 can be as is shown in Scheme 3. MAD derivative 4 or 7 can beconjugated to doxorubicin by formation of hydrazone linkage underanhydrous acidic condition or aqueous acidic conditions. Unconjugatedtherapeutic agent can be removed (e.g. by size exclusion chromatographyor dialyzation) to obtain the pure conjugated MAD (indicated asm-tilmanocept in the scheme below).

Amine-containing therapeutic agents may be conjugated todextran-containing compounds according to Scheme 4. The basic reactionbetween a primary amine and the lactone are shown in Scheme 4.

One of ordinary skill in the art would recognize other ways tosynthesize the compounds of the present invention.

Pharmaceutical Compositions

Embodiments of the invention relate to pharmaceutical compositionscomprising the disclosed compounds and products of disclosed methods.That is, a pharmaceutical composition can be provided comprising aneffective amount of at least one disclosed compound, at least oneproduct of a disclosed method, or a pharmaceutically acceptable salt,solvate, hydrate, or polymorph thereof, and a pharmaceuticallyacceptable carrier. In some embodiments, the invention relates topharmaceutical compositions comprising a pharmaceutically acceptablecarrier and an effective amount of at least one disclosed compound; or apharmaceutically acceptable salt, hydrate, solvate, or polymorphthereof.

In some embodiments, the effective amount is a therapeutically effectiveamount. In certain embodiments, the effective amount is aprophylactically effective amount. In some embodiments, thepharmaceutical composition comprises a compound that is a product of adisclosed method of making.

In some embodiments, the pharmaceutical composition comprises adisclosed compound. In some embodiments, the pharmaceutical compositioncomprises a product of a disclosed method of making.

In certain embodiments, the pharmaceutical composition is used to treata mammal and the mammal can be a human. In some embodiments, the mammalhas been diagnosed with a need for treatment of the disorder prior tothe administering step. In some embodiments, the mammal has beenidentified to be in need of treatment of the disorder.

In certain embodiments, the disclosed pharmaceutical compositionscomprise the disclosed compounds (including pharmaceutically acceptablesalt(s) thereof) as an active ingredient, a pharmaceutically acceptablecarrier, and, optionally, other therapeutic ingredients or adjuvants.The instant compositions include those suitable for oral, rectal,topical, and parenteral (including subcutaneous, intramuscular,intradermal and intravenous) administration, although the most suitableroute in any given case will depend on the particular host, and natureand severity of the conditions for which the active ingredient is beingadministered. The pharmaceutical compositions can be convenientlypresented in unit dosage form and prepared by any of the methods wellknown in the art of pharmacy.

As used herein, the term “pharmaceutically acceptable salts” refers tosalts prepared from pharmaceutically acceptable non-toxic bases oracids. When the compound of the present invention is acidic, itscorresponding salt can be conveniently prepared from pharmaceuticallyacceptable non-toxic bases, including inorganic bases and organic bases.Salts derived from such inorganic bases include aluminum, ammonium,calcium, copper (-ic and -ous), ferric, ferrous, lithium, magnesium,manganese (-ic and -ous), potassium, sodium, zinc and the like salts.Particularly preferred are the ammonium, calcium, magnesium, potassiumand sodium salts. Salts derived from pharmaceutically acceptable organicnon-toxic bases include salts of primary, secondary, and tertiaryamines, as well as cyclic amines and substituted amines such asnaturally occurring and synthesized substituted amines. Otherpharmaceutically acceptable organic nontoxic bases from which salts canbe formed include ion exchange resins such as, for example, arginine,betaine, caffeine, choline, N,N′-dibenzylethylenediamine, diethylamine,2-di ethylaminoethanol, 2-dimethylaminoethanol, ethanolamine,ethylenediamine, N-ethylmorpho line, N-ethylpiperidine, glucamine,glucosamine, histidine, hydrabamine, isopropylamine, lysine,methylglucamine, morpholine, piperazine, piperidine, polyamine resins,procaine, purines, theobromine, triethylamine, trimethylamine,tripropylamine, tromethamine and the like.

As used herein, the term “pharmaceutically acceptable non-toxic acids,”includes inorganic acids, organic acids, and salts prepared therefrom,for example, acetic, benzenesulfonic, benzoic, camphorsulfonic, citric,ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric,isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic,nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric,p-toluenesulfonic acid and the like. Preferred are citric, hydrobromic,hydrochloric, maleic, phosphoric, sulfuric, and tartaric acids.

In practice, the compounds of the invention, or pharmaceuticallyacceptable salts thereof, of this invention can be combined as theactive ingredient in intimate admixture with a pharmaceutical carrieraccording to conventional pharmaceutical compounding techniques. Thecarrier can take a wide variety of forms depending on the form ofpreparation desired for administration, e.g., oral or parenteral(including intravenous). Thus, the pharmaceutical compositions of thepresent invention can be presented as discrete units suitable for oraladministration such as capsules, cachets or tablets each containing apredetermined amount of the active ingredient. Further, the compositionscan be presented as a powder, as lyophilized powder, as granules, as asolution, as a suspension in an aqueous liquid, as a non-aqueous liquid,as an oil-in-water emulsion or as a water-in-oil liquid emulsion. Inaddition to the common dosage forms set out above, the compounds of theinvention, and/or pharmaceutically acceptable salt(s) thereof, can alsobe administered by controlled release means and/or delivery devices. Thecompositions can be prepared by any of the methods of pharmacy. Ingeneral, such methods include a step of bringing into association theactive ingredient with the carrier that constitutes one or morenecessary ingredients. In general, the compositions are prepared byuniformly and intimately admixing the active ingredient with liquidcarriers or finely divided solid carriers or both. The product can thenbe conveniently shaped into the desired presentation.

Thus, the pharmaceutical compositions of this invention can include apharmaceutically acceptable carrier and a compound or a pharmaceuticallyacceptable salt of the compounds of the invention. The compounds of theinvention, or pharmaceutically acceptable salts thereof, can also beincluded in pharmaceutical compositions in combination with one or moreother therapeutically active compounds.

The pharmaceutical carrier employed can be, for example, a solid,liquid, or gas. Examples of solid carriers include lactose, terra alba,sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, andstearic acid. Examples of liquid carriers are sugar syrup, peanut oil,olive oil, and water. Examples of gaseous carriers include carbondioxide and nitrogen.

In preparing the compositions for oral dosage form, any convenientpharmaceutical media can be employed. For example, water, glycols, oils,alcohols, flavoring agents, preservatives, coloring agents and the likecan be used to form oral liquid preparations such as suspensions,elixirs and solutions; while carriers such as starches, sugars,microcrystalline cellulose, diluents, granulating agents, lubricants,binders, disintegrating agents, and the like can be used to form oralsolid preparations such as powders, capsules and tablets. Because oftheir ease of administration, tablets and capsules are the preferredoral dosage units whereby solid pharmaceutical carriers are employed.Optionally, tablets can be coated by standard aqueous or nonaqueoustechniques.

A tablet containing the composition of this invention can be prepared bycompression or molding, optionally with one or more accessoryingredients or adjuvants. Compressed tablets can be prepared bycompressing, in a suitable machine, the active ingredient in afree-flowing form such as powder or granules, optionally mixed with abinder, lubricant, inert diluent, surface active or dispersing agent.Molded tablets can be made by molding in a suitable machine, a mixtureof the powdered compound moistened with an inert liquid diluent.

The pharmaceutical compositions of the present invention comprise acompound of the invention (or pharmaceutically acceptable salts thereof)as an active ingredient, a pharmaceutically acceptable carrier, andoptionally one or more additional therapeutic agents or adjuvants. Theinstant compositions include compositions suitable for oral, rectal,topical, and parenteral (including subcutaneous, intramuscular, andintravenous) administration, although the most suitable route in anygiven case will depend on the particular host, and nature and severityof the conditions for which the active ingredient is being administered.The pharmaceutical compositions can be conveniently presented in unitdosage form and prepared by any of the methods well known in the art ofpharmacy.

Pharmaceutical compositions of the present invention suitable forparenteral administration can be prepared as solutions or suspensions ofthe active compounds in water. A suitable surfactant can be includedsuch as, for example, hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofin oils. Further, a preservative can be included to prevent thedetrimental growth of microorganisms.

Pharmaceutical compositions of the present invention suitable forinjectable use include sterile aqueous solutions or dispersions.Furthermore, the compositions can be in the form of sterile powders forthe extemporaneous preparation of such sterile injectable solutions ordispersions. In all cases, the final injectable form must be sterile andmust be effectively fluid for easy syringability. The pharmaceuticalcompositions must be stable under the conditions of manufacture andstorage; thus, preferably should be preserved against the contaminatingaction of microorganisms such as bacteria and fungi. The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (e.g., glycerol, propylene glycol and liquid polyethyleneglycol), vegetable oils, and suitable mixtures thereof.

Pharmaceutical compositions of the present invention can be in a formsuitable for topical use such as, for example, an aerosol, cream,ointment, lotion, dusting powder, mouth washes, gargles, and the like.Further, the compositions can be in a form suitable for use intransdermal devices. These formulations can be prepared, utilizing acompound of the invention, or pharmaceutically acceptable salts thereof,via conventional processing methods. As an example, a cream or ointmentis prepared by mixing hydrophilic material and water, together withabout 5 wt % to about 10 wt % of the compound, to produce a cream orointment having a desired consistency.

Pharmaceutical compositions of this invention can be in a form suitablefor rectal administration wherein the carrier is a solid. It ispreferable that the mixture forms unit dose suppositories. Suitablecarriers include cocoa butter and other materials commonly used in theart. The suppositories can be conveniently formed by first admixing thecomposition with the softened or melted carrier(s) followed by chillingand shaping in molds.

In addition to the aforementioned carrier ingredients, thepharmaceutical formulations described above can include, as appropriate,one or more additional carrier ingredients such as diluents, buffers,flavoring agents, binders, surface-active agents, thickeners,lubricants, preservatives (including anti-oxidants) and the like.Furthermore, other adjuvants can be included to render the formulationisotonic with the blood of the intended recipient. Compositionscontaining a compound of the invention, and/or pharmaceuticallyacceptable salts thereof, can also be prepared in powder or liquidconcentrate form.

It is understood, however, that the specific dose level for anyparticular patient will depend upon a variety of factors and such adosage amount would be readily ascertainable by the skilled artisan inconsideration of these factors. Such factors include the age, bodyweight, general health, sex, and diet of the patient. Other factorsinclude the time and route of administration, rate of excretion, drugcombination, and the type and severity of the particular diseaseundergoing therapy.

Diagnostic Methods

Diagnostic methods are disclosed for in vivo detection of diseases orconditions using the disclosed compounds.

In certain embodiments, the disclosed compounds include a detectionlabel in addition to the therapeutic agent. As used herein, the term“detectable label or moiety” means an atom, isotope, or chemicalstructure which is: (1) capable of attachment to the carrier molecule;(2) non-toxic to humans or other mammalian subjects; and (3) provides adirectly or indirectly detectable signal, particularly a signal whichnot only can be measured but whose intensity is related (e.g.,proportional) to the amount of the detectable moiety. The signal may bedetected by any suitable means, including spectroscopic, electrical,optical, magnetic, auditory, radio signal, or palpation detection means.

Detection labels include, but are not limited to, fluorescent molecules(a.k.a. fluorochromes and fluorophores), chemiluminescent reagents(e.g., luminol), bioluminescent reagents (e.g., luciferin and greenfluorescent protein (GFP)), metals (e.g., gold nanoparticles), andradioactive isotopes (radioisotopes). Suitable detection labels can beselected based on the choice of imaging method. For example, thedetection label can be a near infrared fluorescent dye for opticalimaging, a Gadolinium chelate for MRI imaging, a radionuclide for PET orSPECT imaging, or a gold nanoparticle for CT imaging.

Detection labels can be selected from, for example, a radionuclide, aradiological contrast agent, a paramagnetic ion, a metal, a fluorescentlabel, a chemiluminescent label, an ultrasound contrast agent, aphotoactive agent, or a combination thereof. Non-limiting examples ofdetectable labels include a radionuclide such as ¹¹⁰In, ¹¹¹In, ¹⁷⁷Lu,¹⁸F, ⁵²Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁸⁶Y, ⁹⁰Y, ⁸⁹Zr, ^(94m)Tc, ⁹⁴Tc^(99m)Tc, ¹²⁰I, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁵⁴-¹⁵⁸Gd, ³²P, ¹¹C, ¹³N, ¹⁵O,¹⁸⁶Re, ¹⁸⁸Re, ⁵¹Mn, ^(52m)Mn ⁵⁵Co, ⁷²As, ⁷⁵Br, ⁷⁶Br, ^(82m)Rb, ⁸³Sr,^(117m)Sn or other gamma-, beta-, or ¹⁸⁶Re, positron-emitters.Paramagnetic ions of use may include chromium (III), manganese (II),iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium(III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II),terbium (III), dysprosium (III), holmium (III) or erbium (III). Metalcontrast agents may include lanthanum (III), gold (III), lead (II) orbismuth (III). Ultrasound contrast agents may comprise liposomes, suchas gas-filled liposomes.

Other suitable labels include, for example, fluorescent labels (such asfluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin,allophycocyanin, o-phthaldehyde, and fluorescamine and fluorescentmetals such as Eu or others metals from the lanthanide series), near IRdyes, quantum dots, phosphorescent labels, chemiluminescent labels orbioluminescent labels (such as luminal, isoluminol, theromaticacridinium ester, imidazole, acridinium salts, oxalate ester, dioxetaneor GFP and its analogs), radio-isotopes, metals, metals chelates ormetallic cations or other metals or metallic cations that areparticularly suited for use in in vivo, in vitro or in situ diagnosisand imaging, as well as chromophores and enzymes (such as malatedehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeastalcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triosephosphate isomerase, biotinavidin peroxidase, horseradish peroxidase,alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase,ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase,glucoamylase and acetylcholine esterase). Other suitable labels includemoieties that can be detected using NMR or ESR spectroscopy. Suchlabeled molecules may, for example, be used for in vitro, in vivo or insitu assays (including immunoassays known per se such as ELISA, RIA, EIAand other “sandwich assays,” etc.) as well as in vivo diagnostic andimaging purposes, depending on the choice of the specific label. Anothermodification may involve the introduction of a chelating group, forexample, to chelate one of the metals or metallic cations referred toabove. Suitable chelating groups, for example, include, withoutlimitation, diethyl-enetriaminepentaacetic acid (DTPA) orethylenediaminetetraacetic acid (EDTA). Yet another modification maycomprise the introduction of a functional group that is one part of aspecific binding pair, such as the biotin(strept)avidin binding pair.Such a functional group may be used to link a disclosed compound to aprotein, polypeptide or chemical compound that is bound to the otherhalf of the binding pair, i.e., through formation of the binding pair.For example, such a conjugated molecule may be used as a reporter, forexample, in a diagnostic system where a detectable signal-producingagent is conjugated to avidin or streptavidin.

Optical Imaging

The disclosed compounds can include a detectable label useful foroptical imaging. A number of approaches can be used for optical imaging.The various methods depend upon fluorescence, bioluminescence,absorption or reflectance as the source of contrast. Fluorophores arecompounds or moieties that absorb energy of a specific wavelength andre-emit energy at a different (but equally specific) wavelength. Incertain embodiments, the detectable label is a near-infrared (NIR)fluorophore. Suitable NIRs include, but are not limited to, VivoTag-S®680 and 750, Kodak X-SIGHT Dyes and Conjugates, DyLight 750 and 800Fluors, Cy 5, Cy 5.5 and 7 Fluors, Alexa Fluor 680 and 750 Dyes, AlexaFluor 688, and IRDye 680 and 800CW Fluors and combinations thereof. Incertain embodiments, Quantum dots, with their photostability and brightemissions, can also be used with optical imaging.

Nuclear Medicine Imaging

The disclosed compounds can include a detectable label (e.g., aradionuclide) useful for nuclear medicine imaging. Nuclear medicineimaging involves the use and detection of radioisotopes in the body.Nuclear medicine imaging techniques include scintigraphy, single photonemission computed tomography (SPECT), and positron emission tomography(PET). In these techniques, radiation from the radioisotopes can becaptured by a gamma camera to form two-dimensional images (scintigraphy)or 3-dimensional images (SPECT and PET).

Radioisotopes that can be incorporated into or attached directly to thedisclosed compounds include, but are not limited to, tritium, ¹¹C, ¹³N,¹⁴C, ¹⁵O, ¹⁸Fl, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁸Ga, ⁷⁶Br, ⁸²Rb, ⁹⁰Y, ^(99m)Tc,¹¹¹In, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁵³Sm, ²⁰¹Tl, ¹⁸⁶Re, ¹⁸⁸Re, ^(117m)Sn and²¹²Bi. In certain embodiments, the radioisotope is attached to adisclosed compound by halogenation. Radionuclides used in PET scanningare typically isotopes with short half-lives. Typical isotopes include¹¹C, ¹³N, ¹⁵O, ¹⁸F, ⁶⁴Cu, ⁶²Cu, ¹²⁴I, ⁷⁶Br, ⁸²Rb and ⁶⁸Ga, with ¹⁸Fbeing the most clinically utilized.

Gamma radiation from radioisotopes can be detected using a gammaparticle detection device. In some embodiments, the gamma particledetection device is a Gamma Finder® device (SenoRx, Irvine Calif.). Insome embodiments, the gamma particle detection device is a Neoprobe® GDSgamma detection system (Dublin, Ohio).

Positron emission tomography is a nuclear medicine imaging techniquewhich produces a three-dimensional image or picture of functionalprocesses in the body. Some agents used for PET imaging provideinformation about tissue metabolism or some other specific molecularactivity. Commonly used agents or potential agents that can be used asdetectable agents include, but are not limited to: ⁶⁴Cudiacetyl-bis(N⁴-methylthiosemicarbazone); ¹⁸F-fluorodeoxyglucose (FDG);¹⁸F-fluoride; 3′-deoxy-3′-[¹⁸F]fluorothymidine (FLT);¹⁸F-fluoromisonidazole; Gallium; Technetium-99m; and Thallium.Radiopaque diagnostic agents may be selected from barium compounds,gallium compounds, and/or thallium compounds. A wide variety offluorescent labels are known in the art, including but not limited tofluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin,allophycocyanin, o-phthaldehyde and fluorescamine. Chemiluminescentlabels of use may include luminol, isoluminol, an aromatic acridiniumester, an imidazole, an acridinium salt or an oxalate ester.

A number of trivalent metal radionuclides have physical propertiessuitable for radioisotope imaging (e.g., indium-111 (¹¹¹In)gallium-67/68 (^(67/68)Ga) and yttrium-86 (⁸⁶Y)) or for targetedradionuclide therapy (e.g., ⁹⁰Y and lutetium-177 (¹⁷⁷Lu)).Diethylenetriaminepentaacetic acid (DTPA) and/or1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA; CAS60239-18-1) can be used (see Choe and Lee, 2007, Current PharmaceuticalDesign, 13:17-31; Li et al., 2007, J. Nuclear Medicine, “⁶⁴Cu-LabeledTetrameric and Octameric RGD Peptides forSmall-Animal PET of Tumor avb3Integrin Expression”, 48:1162-1171; Nahrendorf et al, 2009, JACCCardiovasc. Imaging, 2:10:1213-1222; Li et al., 2009, Mol. Cancer Ther.,8:5:1239-1249; Yim et al., 2010, J. Med. Chem., 53:3944-3953; Dijkgraafet al., 2010, Eur. J. Nucl. Med. Mol. Imaging, published online 21 Sep.2010; U.S. patent application Ser. No. 10/792,582; Dransfield et al.,U.S. Pat. Pub. Nos. US 2010/0261875; U.S. Pat. No. 7,666,979). Of themetals mentioned, the DOTA complexes are more thermodynamically andkinetically stable than the DTPA complexes (see Sosabowski et al.,Nature Protocols 1, 972-976 (2006) and Leon-Rodriguez et al.,Bioconjugate chemistry, Jan. 3, 2008; 19(2):391-402).

Magnetic Resonance Imaging

The disclosed compounds can be detected via magnetic resonance imaging.MRI has the advantages of having very high spatial resolution and isvery adept at morphological imaging and functional imaging. MRIgenerally has a sensitivity of around 10⁻³ mol/L to 10⁻⁵ mol/L.Improvements to increase MR sensitivity include hyperpolarization byincreasing magnetic field strength, optical pumping, or dynamic nuclearpolarization. There are also a variety of signal amplification schemesbased on chemical exchange that increase sensitivity.

Chelating Agents

In some embodiments, a chelating agent may be attached to orincorporated into a disclosed compound, and used to chelate atherapeutic or diagnostic agent, such as a radionuclide. Exemplarychelators include but are not limited to DTPA (such as Mx-DTPA), DOTA,TETA, NETA or NOTA.

Useful chelators include, but are not limited to, DTPA, DO3A, DOTA,EDTA, TETA, EHPG, HBED, NOTA, DOTMA, TETMA, PDTA, TTHA, LICAM, HYNIC,and MECAM. HYNIC is particularly useful for chelating Tc99, anotherimaging agent of the invention.

Actions Based on Imaging and Identifications

The disclosed methods include the determination, identification,indication, correlation, diagnosis, prognosis, etc. (which can bereferred to collectively as “identifications”) of subjects, diseases,conditions, states, etc. based on imagings, measurements, detections,comparisons, analyses, assays, screenings, etc. For example, thedisclosed imaging methods allow identification of patients, organs,tissues, etc. having cancer cells, metastasized cancer cells, cancercells beyond tumor margins, etc. Such identifications are useful formany reasons. For example, and in particular, such identifications allowspecific actions to be taken based on, and relevant to, the particularidentification made. For example, diagnosis of a particular disease orcondition in particular subjects (and the lack of diagnosis of thatdisease or condition in other subjects) has the very useful effect ofidentifying subjects that would benefit from treatment, actions,behaviors, etc. based on the diagnosis. For example, treatment for aparticular disease or condition in subjects identified is significantlydifferent from treatment of all subjects without making such anidentification (or without regard to the identification). Subjectsneeding or that could benefit from the treatment will receive it andsubjects that do not need or would not benefit from the treatment willnot receive it.

Accordingly, also disclosed herein are methods comprising takingparticular actions following and based on the disclosed identifications.For example, disclosed are methods comprising creating a record of anidentification (in physical—such as paper, electronic, or other—form,for example). Thus, for example, creating a record of an identificationbased on the disclosed methods differs physically and tangibly frommerely performing a imaging, measurement, detection, comparison,analysis, assay, screen, etc. Such a record is particularly substantialand significant in that it allows the identification to be fixed in atangible form that can be, for example, communicated to others (such asthose who could treat, monitor, follow-up, advise, etc. the subjectbased on the identification); retained for later use or review; used asdata to assess sets of subjects, treatment efficacy, accuracy ofidentifications based on different imagings, measurements, detections,comparisons, analyses, assays, screenings, etc., and the like. Forexample, such uses of records of identifications can be made, forexample, by the same individual or entity as, by a different individualor entity than, or a combination of the same individual or entity as anda different individual or entity than, the individual or entity thatmade the record of the identification. The disclosed methods of creatinga record can be combined with any one or more other methods disclosedherein, and in particular, with any one or more steps of the disclosedmethods of identification.

As another example, disclosed are methods comprising making one or morefurther identifications based on one or more other identifications. Forexample, particular treatments, monitorings, follow-ups, advice, etc.can be identified based on the other identification. For example,identification of a subject as having a disease or condition with a highlevel of a particular component or characteristic can be furtheridentified as a subject that could or should be treated with a therapybased on or directed to the high level component or characteristic. Arecord of such further identifications can be created (as describedabove, for example) and can be used in any suitable way. Such furtheridentifications can be based, for example, directly on the otheridentifications, a record of such other identifications, or acombination. Such further identifications can be made, for example, bythe same individual or entity as, by a different individual or entitythan, or a combination of the same individual or entity as and adifferent individual or entity than, the individual or entity that madethe other identifications. The disclosed methods of making a furtheridentification can be combined with any one or more other methodsdisclosed herein, and in particular, with any one or more steps of thedisclosed methods of identification.

As another example, disclosed are methods comprising treating,monitoring, following-up with, advising, etc. a subject identified inany of the disclosed methods. Also disclosed are methods comprisingtreating, monitoring, following-up with, advising, etc. a subject forwhich a record of an identification from any of the disclosed methodshas been made. For example, particular treatments, monitorings,follow-ups, advice, etc. can be used based on an identification and/orbased on a record of an identification. For example, a subjectidentified as having a disease or condition with a high level of aparticular component or characteristic (and/or a subject for which arecord has been made of such an identification) can be treated with atherapy based on or directed to the high level component orcharacteristic. Such treatments, monitorings, follow-ups, advice, etc.can be based, for example, directly on identifications, a record of suchidentifications, or a combination. Such treatments, monitorings,follow-ups, advice, etc. can be performed, for example, by the sameindividual or entity as, by a different individual or entity than, or acombination of the same individual or entity as and a differentindividual or entity than, the individual or entity that made theidentifications and/or record of the identifications. The disclosedmethods of treating, monitoring, following-up with, advising, etc. canbe combined with any one or more other methods disclosed herein, and inparticular, with any one or more steps of the disclosed methods ofidentification.

Methods of Treatment

Methods of treating or preventing diseases or disorders are providedusing the disclosed compounds. The disclosed compounds can be used fortargeting CD206 high expressing cells and/or for targeting ofmacrophages for treatment of intracellular pathogens. Some embodimentsas disclosed herein by target cells that do not express CD206. Thedisclosed compounds can be used to target tumor-associated macrophages.Some embodiments relate to methods of treating viral infectionsincluding flaviviridae viruses such as, for example but withoutlimitation, yellow fever, dengue virus, and zika virus. Some embodimentsrelate to treatment of syndromes or symptoms of these, such as but notlimited to, Guillan-Barre syndrome.

Tilmanocept and its equivalents are discussed in PCT/US2015/041036, theentirety of which is incorporated herein by reference.

Compositions disclosed herein can be used to treat and/or diagnose Zikavirus, or other viruses as disclosed herein such as yellow fever, Denguevirus, and other flaviviridae viruses. Compositions disclosed herein canbe administered to a subject to prevent acquisition of Zika virus.Compositions disclosed herein can be administered to a subjectexhibiting symptoms of Zika virus, to treat Zika virus and/or thesymptoms of Zika virus. A subject can be exposed to compositionsdisclosed herein prior to infection to prevent or ameliorate infection.

In certain embodiments, agents, compounds and/or compositions comprisetilmanocept (TIL, dextran 3-[(2-aminoethyl)thio]propyl17-carboxy-10,13,16-tris(carboxymethyl)-8-oxo-4-thia-7,10,13,16-tetraazaheptadec-1-yl-3-[[2-[[1-imino-2-(D-mannopyranosylthio)ethyl]amino]ethyl]-thio]propyl ether complexes), manocept [(MAN,17) tilmanocept sans DTPA],MAN-doxorubicin (MAN,17-DOX,5), and/or MAN-dexamethasone (MAN,17-DEX,5).

Compositions disclosed herein, such as tilmanocept and/or manocept alongwith their congeners, can be used as antivirals against Zika virus,Dengue, or other Flaviviridae family viruses. In certain embodiments,compositions described herein can be administered prior to conceiving achild to prevent the transmission of Zika virus, Dengue, or otherFlavivrida family viruses. In some embodiments, compositions describedherein can be administered to males and/or females to prevent or reducetransmission of Zika virus, Dengue, or other Flavivrida family viruses.

Macrophages make endosomes when they take up compositions describedherein and the compositions described herein may not be degraded duringthis uptake. As discussed herein, Flaviviruses have a (+) sense RNAgenome and replicate in the cytoplasm of the host cells. In certainembodiments, this replication may occur in macrophages, thus providing away for tilmanocept compositions to deliver therapeutic agents to treatand/or prevent Zika and other flaviruses.

Without being bound by theory, human macrophages may exhibit nocytopathology from Zika viral infection. In certain embodiments,tilmanocept may be used to treat and/or prevent embryo infection whereinsuch an infection has occurred via transmigrating macrophages inplacenta. In some embodiments, tilmanocept compositions may interactwith such macrophages and deliver a therapeutic agent to treat and/orprevent embryonic Zika virus. In such embodiments, tilmanocept can treatand/or prevent birth defects caused by Zika virus, such as microcephaly.

In one experiment, Vero cells are used to test the effectiveness oftilmanocept in directing a therapeutic agent effective for treating,preventing, eliminating, and/or ameliorating Zika virus. Embodiments oftilmanocept as described herein can comprise therapeutic agents asdescribed herein for curing, ameliorating, preventing, and/or treatingZika virus and/or the symptoms of Zika virus along with otherflaviviridae viruses as discussed herein. In certain embodiments wheretilmanocept is used to cure, ameliorate, prevent, and/or treat Zikavirus, the tilmanocept may comprise a therapeutic agent such asdoxorubicin. In certain embodiments, tilmanocept is administered to apatient to cure, ameliorate, prevent, kill, eliminate, and/or treatsymptoms caused by Zika virus. To cure, ameliorate, prevent, and/ortreat Zika virus, tilmanocept can be administered to a patient asdescribed herein. In some embodiments, tilmanocept comprising a Zikavirus therapeutic agent can be administered as a local intradermalinjection. In certain embodiments, tilmanocept comprising a Zika virustherapeutic agent can be administered intravenously.

Tilmanocept as well as other related carrier molecules described in the'990 Patent, as well as other carrier molecules based on a dextranbackbone, bind exclusively to the mannose receptor protein CD206 foundon the surface of macrophages and certain other cells (e.g., Kaposi'ssarcoma spindle cells) when administered to mammals or when contactedwith CD206 high expressing cells ex vivo. No other receptors arebelieved to bind or transduce these carrier molecules, even though thereare numerous other mannose receptors found in mammals. CD206 is a C-typelecithin binding protein found on the surface of macrophages and certainother types of cells. The finding that the CD206 protein, found forexample on the surface of macrophages, is the sole gateway fortilmanocept binding in mammalian patients means that a MAD carriermolecule (as well as related carrier molecules) can be used as the basisfor preparing a variety of therapeutically and diagnostically effectivemolecular species for use in the diagnosis and/or treatment ofmacrophage related diseases and other diseases mediated by CD206 highexpressing cells.

The disclosed compounds can include therapeutic agents including, butnot limited to, cytotoxic agents, anti-angiogenic agents, pro-apoptoticagents, antibiotics, hormones, hormone antagonists, chemokines, drugs,prodrugs, toxins, enzymes, or other agents. The disclosed compounds caninclude chemotherapeutic agents; antibiotics; immunological adjuvants;compounds useful for treating tuberculosis; steroids; nucleotides;peptides; or proteins.

In certain embodiments, the compounds include an antimicrobial drugselected from the group comprising or consisting of: an antibiotic; ananti-tuberculosis antibiotic (such as isoniazid, ethambutol); ananti-retroviral drug, for example an inhibitor of reverse transcription(such as zidovudin) or a protease inhibitor (such as indinavir); drugswith effect on leishmaniasis (such as Meglumine antimoniate), or anycombination thereof. In certain embodiments, the compounds include ananti-microbial active, such as amoxicillin, ampicillin, tetracyclines,aminoglycosides (e.g., streptomycin), macrolides (e.g., erythromycin andits relatives), chloramphenicol, ivermectin, rifamycins and polypeptideantibiotics (e.g., polymyxin, bacitracin) and zwittermicin. In certainembodiments, the compounds include an active selected from isoniazid,doxorubicin, streptomycin, and tetracycline, or any combination thereof.The disclosed compounds can be used, for example, to treat Tuberculosis,Staphylococcus, Streptococcus, yeast, Serratia. E. coli, and Pseudomonasaeruginosa, Zika, and/or dengue infections.

In certain embodiments, the disclosed compounds advantageously haveefficacy in the treatment of a condition, disease, or disorder caused bya micro-organism, for example, a condition, disease, or disorderincluding, for example, Zika and dengue.

One of ordinary skill in the art will appreciate that various kinds ofmolecules and compounds (e.g., therapeutic agents, detection labels, andcombinations thereof) can be delivered to a cell or tissue using thedisclosed compounds.

Administration

The disclosed compounds can be administered via any suitable method. Thedisclosed compounds can be administered parenterally into the parenchymaor into the circulation so that the disclosed compounds reach targettissues (e.g., where viral-infected cells may be located). The disclosedcompounds can be administered intravenously. In still other embodiments,the disclosed compounds can be administered intraperitoneally,intramuscularly, subcutaneously, intracavity, or transdermally.

Parenteral administration of the compounds, if used, is generallycharacterized by injection. Injectables can be prepared in conventionalforms, either as liquid solutions or suspensions, solid forms suitablefor solution of suspension in liquid prior to injection, or asemulsions. A revised approach for parenteral administration involves useof a slow release or sustained release system such that a constantdosage is maintained.

EXAMPLES Example 1. Cy3-Tilmanocept Binding to Human Macrophages

A quantity of peripheral blood mononuclear cell (PBMC)s consisting oflymphocytes or macrophages was cultured for 5 days to enable bloodmonocytes to differentiate into macrophages (human monocyte-derivedmacrophages, or “MDMs”), and then pre-treated with or without unlabeled(cold) tilmanocept. Next, the cells were incubated with varyingconcentrations (1.25, 2.5, 5.0, 10 and 20 μg/mL) of Cy3-labeledtilmanocept (Cy3-tilmanocept). Binding to PBMC cell populations wasanalyzed by flow cytometry by gating separately for macrophages andlymphocytes. The resulting data showed that Cy3-tilmanocept bindsspecifically to the macrophage population in a dose-dependent manner, asshown in FIG. 1A. FIG. 1A depicts fluorescence-activated cell sorting(“FACS”) analysis of PBMCs, focusing on macrophages and lymphocytes. Forthe macrophages that were pre-treated with cold tilmanocept (100-foldexcess), the binding of Cy3-tilmanocept was nearly abolished even at thehighest concentrations, as shown in FIG. 1B (FACS analysis showinginhibition of tilmanocept-Cy3 binding to macrophages in presence ofunlabeled tilmanocept **P<0.005).

To corroborate these findings, MDMs were treated in monolayer culture ina similar way, and fluorescence confocal microscopy experiments wereperformed. The binding of Cy3-tilmanocept to macrophages was readilyapparent and this binding was nearly abolished for macrophages that werepre-treated with cold tilmanocept, as seen in FIG. 1C. Depicted data isrepresentative of two independent experiments, each performed induplicate, and the results were consistent with receptor-mediatedbinding of Cy3-tilmanocept to macrophages. The upper and lower leftimages in FIG. 1C depict confocal microscopy representative images(magnification: 120×) which show binding (upper left) and inhibition ofbinding (lower left) of Cy3-tilmanocept to macrophages in the absence orpresence of tilmanocept with no fluorophore, respectively. The grayregions indicate macrophage nuclei, and the white portions indicateCy3-tilmanocept. The upper and lower right images in FIG. 1C are DICimages which show the individual cell structure of the adjacentfluorescent images (to the left of each DIC image). “DIC” isDifferential Interference Contrast (phase contrast microscopy).

Example 2. Co-Localization of Tilmanocept with the CD206 MannoseReceptor on Human Macrophages

MDM monolayers were incubated with Cy3-tilmanocept for 10 minutes, fixedwith paraformaldehyde, incubated with anti-MR primary Ab, and stainedwith Alexa Fluor 488-conjugated secondary Ab. The monolayers were thenanalyzed by confocal microscopy. FIG. 2 illustrates representativeconfocal images (magnification: 160×) showing expression of the CD206 MR(FIG. 2A), Cy3-tilmanocept binding by the macrophage (FIG. 2B), andco-localization between the MR and Cy3-tilmanocept in both confocal andphase contrast images (FIGS. 2C and 2D). The results shown arerepresentative of three independent experiments.

Example 3. Binding of Cy3-Tilmanocept to Macrophages Infected withTuberculosis

Human monocyte-derived macrophages in monolayer culture that make up thecomponents of the TB granulomas were infected with a GFP-expressing M.tuberculosis which was internalized by macrophages (GFP=greenfluorescent protein). The infected cells were then exposed toCy3-tilmanocept which had been labeled with cyanine (Cy3) dye, andanalyzed by confocal microscopy. Thus, FIG. 3 demonstrates that theCy3-tilmanocept binds to, and is internalized by the macrophages.

Example 4. Using Tilmanocept for Zika Virus-Infected VEROS Cells

African Green Monkey kidney epithelial cells (Vero; ATCC #CCL-81) and aclone of standard Vero cells (E6; ATCC #CRL-1586) are grown in minimalessential medium (MEM; Gibco, Carlsbad, Calif.) supplemented with 10%fetal bovine serum (FBS; Hyclone, Logan, Utah), 2 mM L-glutamine, 1.5g/l sodium bicarbonate, 100 U/ml of penicillin, 100 μg/ml ofstreptomycin, and incubated at 37° C. in 5% CO₂.

Human monocyte-derived macrophage isolation—Human peripheral bloodmonocyte-derived macrophages (human PCMBs, which can also be referred toas HPBM) may be purchased from appropriate commercial vendors.Alternatively, human PBMCs is isolated from heparinized blood fromhealthy donors (from Red Cross buffy coat preparations—upon request) ona FicollHypaque (Amersham, Pittsburgh, Pa.) cushion and cultured inTeflon wells in RPMI 1640+20% autologous serum for 5 days at 37° C., 5%CO₂ (19), during which time monocytes differentiate intomonocyte-derived macrophages (MDMs). Such cells are used in suspensionor as purified MDMs in monolayer culture. Experiments are performed induplicate or triplicate wells.

Zika or Dengue virus screens and titrations for virus quantification canbe completed by plaque assay on Vero cell cultures. Duplicate wells areinfected with 0.1 ml aliquots from serial 10-fold dilutions in growthmedia and virus is adsorbed for one hour. Following incubation, theinoculum is removed, and monolayers are overlaid with 3 ml containing a1:1 mixture of 1.2% oxoid agar and 2×DMEM (Gibco, Carlsbad, Calif.) with10% (vol/vol) FBS and 2% (vol/vol) penicillin/streptomycin. Cells areincubated at 37° C. in 5% CO₂ for up to four days for plaquedevelopment. Cell monolayers then are stained with 3 ml of overlaycontaining a 1:1 mixture of 1.2% oxoid agar and 2×DMEM with 2% (vol/vol)FBS, 2% (vol/vol) penicillin/streptomycin, and 0.33% neutral red(Gibco). Cells are incubated overnight at 37° C. and plaques arecounted.

TIL and MAN,17 congeners as well as fluorescent congeners are suppliedby Macrophage Therapeutics (MT; a subsidiary of NavideaBiopharmaceuticals, Dublin, Ohio). The test compounds and theirspecifications (the K_(d) range for the agents is 3×10⁻¹¹ to 1×10⁻¹⁰ M;molecular weights, mole ratios of core molecules to active conjugateddrug, e.g. dextran/mannose to doxorubicin) are supplied by MT. Agentsmay be solubilized in isotonic saline or PBS to produce stock solutionsas may be used in the blocking, inhibition, or fate evaluations.Concentrations of the MAN,17 or TIL or other congeners can be based on amolecular weight of 20 kDa. Anticipated starting concentrations will bein the about 10 nM to about 1 μM range.

A viral infectivity inhibition assay is performed to measure theantiviral efficacy of TIL and MAN,17 congeners in cell culture.

Vero cells or human macrophages are seeded in 24-well plates(approximately 2×10⁴ cells per well) and incubated without the presenceof a drug for 24 hours to form a confluent monolayer. Followingincubation, the medium is aspirated from the wells and replaced with 200μl of fresh designated medium containing 500 nM of the test compound(three wells per compound), which is inoculated with Zika virus at amultiplicity of infection (MOI) that can be determined prior to the timethe test compounds are added. As a negative control, vehicle is added tovirus-infected and mock-infected cells at a final concentration of 0.5%(v/v). Additionally, MAN,17 is used as a negative control. Culturemedium is monitored for 5 days post-infection (p.i.) to yield a 70-90%cytopathic effect (CPE) in virus control wells using a microscopeequipped with a camera. To quantify the CPE, culture media is collectedat the end of the experiment (i.e., day 5 p.i.) and cell death isdetermined using the CytoTox 96 Non-Radioactive Cytotoxicity Assay(Promega; Madison, Wis.) according to the Manufacturer's instructions.

Viral titers are determined by plaque assay and expressed as PFU/m. Fordose-response studies, Vero cell monolayers can be cultured with 200 μlof medium containing the test compounds over the concentration range,and Zika virus at an MOI of 0.01. Drug addition and virus infection canbe done at the same time with medium change time to be determined. Themedium is collected from the wells at 2 and 3 days p.i., the viraltiters can be determined by plaque assay and can be used to constructZika Virus dose-response curves. The dose-response curves on day 2 p.i.can be used to estimate the 50% effective concentration (EC50). Tomeasure the compound-induced inhibition of viral surface antigenexpression, a cell-based flavivirus immunostaining assay is performed asdescribed herein. For determination of nucleoside analogue cytotoxicity,a colorimetric assay utilizing Dojindo's highly water-solubletetrazolium salt (Cell Counting Kit-8, Dojindo Molecular Technologies;Rockville, Md.) and CytoTox 96 Non-Radioactive Cytotoxicity Assay(Promega; Madison, Wis.) can be utilized. The concentration of compoundthat reduced cell viability by 50% is considered as the 50% cytotoxicconcentration (CC50).

Tilmanocept fate evaluation can be based on the use of Til-Alexa 488 orTil-Cy5 fluorescent congeners. The use of these agents in tracking thefate of tilmanocept can provide binding, internalization and blockingpotential interactions with chosen cell models.

Zika virus generally causes a benign disease, but in some patients theinfection can manifest as myelitis or meningoencephalitis, or cantrigger Guillain-Barré syndrome, a severe neurological disordercharacterized by progressive muscle weakness that can result inrespiratory failure. No effective therapy for Zika virus infection isavailable at present.

Dengue virus—Infection with any of the dengue serotypes may beasymptomatic in the majority of cases or may result in a wide spectrumof clinical symptoms, ranging from a mild flu-like syndrome (known asdengue fever [DF]) to the most severe forms of the disease, which arecharacterized by coagulopathy, increased vascular fragility, andpermeability (dengue hemorrhagic fever [DHF]). The latter may progressto hypovolemic shock (dengue shock syndrome [DSS]). Like Zika virus, noeffective therapy for Dengue virus infection is available at present.

Vero cells infected with Zika virus showed an unexpected 99.9% reductionafter administering tilmanocept comprising a therapeutic agent.Doxorubicin is used as a therapeutic agent. In an example, prior toadministration there was 3×10⁶ Zika virions/mL and after administrationof tilmanocept comprising doxorubicin to a subject, this unexpectedlywent down to 2×10³ Zika virions/mL. Plaque assays are prepared showingresults, an example of which is shown as FIG. 4. FIG. 4 shows theresults of administration of (1) a control containing phosphate-bufferedsaline (PBS); (2) 5 μM doxorubicin (Dox 5 μM); 1 μM doxorubicin (Dox 1μM); 5 μM tilmanocept containing 17 mannoses (Man17 5 μM); 1 μMtilmanocept containing 17 mannoses (Man17 1 μM); 5 μM dexamethasone (Dex5 μM); 1 μM dexamethasone (Dex 1 μM); 5 μM tilmanocept containing 17mannoses and dexamethasone (ManDex 5 μM); and 1 μM tilmanoceptcontaining 17 mannoses and dexamethasone (ManDex 1 μM). FIG. 4 shows thesuperior and unexpected results of use of tilmanocept in conjunctionwith a therapeutic agent (e.g., dexamethasone) of reducing Zikainfection in VERO cells. ManDex 5 μM and ManDex 1 μM showedsignificantly improved results over administration of dexamethasonealone. Additional results are also shown in FIG. 5. FIG. 5 shows thepercent of infectivity of VERO cells after administration of thesolutions discussed with respect to FIG. 4. Similarly unexpectedlyimproved results were achieved via administration of ManDex 5 μM andManDex 1 μM, which showed a dramatically improved reduction in Zikainfectivity. Tilmanocept administered without a therapeutic agent had noeffect. Free drug administration had very little effect. Thisdemonstrates the unexpected results that tilmanocept and derivativesthereof, in combination with a therapeutic agent, can be effective insubstantially reducing the amount of virus present in a subject alongwith adequately targeting cells infected with Zika virus.

Vero cells have a receptor that has lower affinity than that ofmacrophages (i.e., no CD206). As mentioned above, administration oftilmanocept showed a 99.9% reduction of Zika virus. This demonstratesthe unexpected and superior results of tilmanocept to show that it hasaffinity for multiple receptors. Tilmanocept can have affinity to humandendritic cells, human skin fibroblasts, human and placentalmacrophages, and can deliver therapeutic agents to these cells if theyare infected with Zika virus.

Example 5. Administration of Tilmanocept Virus to Human Cells Infectedwith Zika Virus

Human activated macrophages were infected with the Zika virus understandard conditions using methods known to those of skill in the art.Those macrophages were cultured and then tilmanocept constructscontaining therapeutic agents were administered and then compared to acontrol group that received no tilmanocept. Three tilmanocept constructswere administered: (1) a tilmanocept containing 16 mannoses anddexamethasone (MANDEX16); (2) a tilmanocept containing 27 mannoses anddexamethasone (MANDEX27); and (3) a tilmanocept containing 27 mannosesand doxorubicin (MANDOX27). Each of these was administered to themacrophage cultures at increasing doses as is shown in FIG. 6. FIG. 6shows the superior and unexpected results of the reduction of infectionamounts achieved after administration of MANDOX27 as follows:

TABLE 1 MANDEX16 MANDEX27 MANDOX27 % Infection % Infection % InfectionApproximate Remaining Remaining Remaining Dosage after after afterAmount administration administration administration 200 nM 0 0 ~55 400nM 0 0 ~40 800 nM 0 0 ~78  2 μM 0 0 ~82  4 μM 0 0 ~90  8 μM 100 100 100Table 1 shows that the administration of MANDOX 27 resulted in a reducedinfection amount wherein that reduction increased with increases indosage rates until 100% of the infection was reduced. The infectionreduction was measured using methods known to those of skill in the art.Without being bound by theory, it is believed that the sudden reductionfrom zero to 100% shown by the administration of 8 μM MANDEX16 andMANDEX27 was a result in total cellular death as a result ofcytotoxicity.

Although the foregoing description is directed to the preferredembodiments of the invention, it is noted that other variations andmodifications will be apparent to those skilled in the art, and may bemade without departing from the spirit or scope of the invention.Moreover, features described in connection with one embodiment of theinvention may be used in conjunction with other embodiments, even if notexplicitly stated above.

What is claimed is:
 1. A method of treating a disease comprisingadministering to a subject in need thereof an effective amount of acompound of Formula (II):

wherein each X is independently H, L₁-A, or L₂-R; each L₁ and L₂ areindependently linkers; each A independently comprises a therapeuticagent or a detection label or H; each R independently comprises a CD206targeting moiety or H; and n is an integer greater than zero; andwherein at least one R is a CD206 targeting moiety and at least one A isa chemotherapeutic agent wherein the disease is selected from ZikaVirus, Yellow Fever, and Dengue Fever.
 2. The method of claim 1, whereina linker is used to attach the one or more CD206 targeting moietiesand/or one or more chemotherapeutic agent.
 3. The method of claim 1,wherein at least one L₁ comprises a degradable linker.
 4. The method ofclaim 1, wherein at least one L₁ comprises a hydrolysable linker.
 5. Themethod of claim 1, wherein at least one L₁ comprises an acid-sensitivelinker.
 6. The method of claim 1, wherein treatment is effective throughbinding of the compound to the CD206 expressing cells of the subject andnot through binding to the virus.
 7. The method of claim 1, whereinadministration of the compound treats the disease and decreases diseasecausing virus through binding of the subject's CD206 expressing cells.8. The method of claim 1, wherein the chemotherapeutic agent isdoxorubicin or dexamethasone.
 9. A method of targeting zika virus bytargeting macrophages comprising administering to a subject in needthereof an effective amount of a compound of Formula (II):

wherein each X is independently H, L₁-A, or L₂-R; each L₁ and L₂ areindependently linkers; each A independently comprises a therapeuticagent or a detection label or H; each R independently comprises a CD206targeting moiety or H; and n is an integer greater than zero; andwherein at least one R is a CD206 targeting moiety and at least one A isa chemotherapeutic agent to treat the disease and decrease the amount ofdisease causing virus.
 10. The method of claim 9, wherein administrationof the compound treats the disease and decreases disease causing virusthrough binding of the subject's CD206 expressing cells.
 11. The methodof claim 9, wherein the chemotherapeutic agent is doxorubicin ordexamethasone.
 12. A method of treating a disease caused by infection bya flaviviridae family virus comprising administering to a subject inneed thereof an effective amount of a compound of Formula (II):

wherein each X is independently H, L₁-A, or L₂-R; each L₁ and L₂ areindependently linkers; each A independently comprises a therapeuticagent or a detection label or H; each R independently comprises a CD206targeting moiety or H; and n is an integer greater than zero; andwherein at least one R is a CD206 targeting moiety and at least one A isdoxorubicin or dexamethasone.